Methods in molecular biology vol 1587 telomeres and telomerase methods and protocols

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Methods in molecular biology vol 1587 telomeres and telomerase methods and protocols

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Methods in Molecular Biology 1587 Zhou Songyang Editor Telomeres and Telomerase Methods and Protocols Third Edition Methods in Molecular Biology Series Editor John M. Walker School of Life and Medical Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB, UK For further volumes: http://www.springer.com/series/7651 Telomeres and Telomerase Methods and Protocols Third Edition Edited by Zhou Songyang Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX, USA Editor Zhou Songyang Department of Biochemistry and Molecular Biology Baylor College of Medicine Houston, TX, USA ISSN 1064-3745     ISSN 1940-6029 (electronic) Methods in Molecular Biology ISBN 978-1-4939-6891-6    ISBN 978-1-4939-6892-3 (eBook) DOI 10.1007/978-1-4939-6892-3 Library of Congress Control Number: 2017932796 © Springer Science+Business Media LLC 2002, 2011, 2017 This work is subject to copyright All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed The use of general descriptive names, registered names, trademarks, service marks, etc in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Printed on acid-free paper This Humana Press imprint is published by Springer Nature The registered company is Springer Science+Business Media LLC The registered company address is: 233 Spring Street, New York, NY 10013, U.S.A Preface In 2009, the Nobel Prize in Physiology or Medicine was awarded to Drs Elizabeth H. Blackburn, Carol W. Greider, and Jack W. Szostak for their pioneering work on telomeres and telomerase, nearly 40 years after the first identification of telomeres Our knowledge of the telomerase and how telomeres are maintained has continued to grow, thanks in no small part to the ever-expanding tools and platforms that are available to investigators It is clear that telomere maintenance is critically linked to cell growth, proliferation, aging, and diseases such as cancer Active investigations are underway to untangle the complex signaling events that lead from telomere dysfunction to premature aging and carcinogenesis In the second volume of Telomeres and Telomerase book (MiMB Vol 735), a variety of assays were presented that allowed investigators to query the activity of telomerase, function of telomere proteins, and the responses of the telomere DNA. Further advances in technology have equipped us with new and improved assays that enable us to ask fundamental questions of telomere regulation in diverse model systems This volume aims to expand the scope further, incorporating some of the newest technologies in the field This combination of genetic, proteomic, genomic, biochemical, and molecular approaches will afford us unprecedented insight into the complex protein interaction networks at work on the telomere chromatin, and the detailed information regarding telomere dynamics in response to stress or stimuli These protocols are detailed and easy to follow It is our belief that this work will prove useful and informative Houston, TX , USA Zhou Songyang v Contents Preface v Contributors ix   Introduction to Telomeres and Telomerase Zhou Songyang   Analysis of Average Telomere Length in Human Telomeric Protein Knockout Cells Generated by CRISPR/Cas9 Jun Xu, Zhou Songyang, Dan Liu, and Hyeung Kim   Telomere Length Analysis by Quantitative Fluorescent in Situ Hybridization (Q-FISH) Isabelle Ourliac-Garnier and Arturo Londoño-Vallejo   Telomere Strand-Specific Length Analysis by Fluorescent In Situ Hybridization (Q-CO-FISH) Isabelle Ourliac-Garnier and Arturo Londoño-Vallejo   Telomere G-Rich Overhang Length Measurement: DSN Method Yong Zhao, Jerry W Shay, and Woodring E Wright   Telomere G-Overhang Length Measurement Method 2: G-Tail Telomere HPA Hidetoshi Tahara   Telomere Terminal G/C Strand Synthesis: Measuring Telomerase Action and C-Rich Fill-In Yong Zhao, Jerry W Shay, and Woodring E Wright   Analysis of Yeast Telomerase by Primer Extension Assays Min Hsu and Neal F Lue   Assessing Telomerase Activities in Mammalian Cells Using the Quantitative PCR-Based Telomeric Repeat Amplification Protocol (qTRAP) Shuai Jiang, Mengfan Tang, Huawei Xin, and Junjiu Huang 10 Telomeres and NextGen CO-FISH: Directional Genomic Hybridization (Telo-dGH™) Miles J McKenna, Erin Robinson, Edwin H Goodwin, Michael N Cornforth, and Susan M Bailey 11 Visualization of Human Telomerase Localization by Fluorescence Microscopy Techniques Eladio Abreu, Rebecca M Terns, and Michael P Terns 12 Cytogenetic Analysis of Telomere Dysfunction Rekha Rai, Asha S Multani, and Sandy Chang 13 Probing the Telomere Damage Response Rekha Rai and Sandy Chang vii 15 29 41 55 63 71 83 95 103 113 127 133 viii Contents 14 Induction of Site-Specific Oxidative Damage at Telomeres by Killerred-Fused Shelretin Proteins Rong Tan and Li Lan 15 Using Protein-Fragment Complementation Assays (PCA) and Peptide Arrays to Study Telomeric Protein-Protein Interactions Wenbin Ma, Ok-hee Lee, Hyeung Kim, and Zhou Songyang 16 In Vitro Preparation and Crystallization of Vertebrate Telomerase Subunits Jing Huang, Christopher J Bley, Dustin P Rand, Julian J.L Chen, and Ming Lei 17 Human Telomeric G-Quadruplex Structures and G-Quadruplex-Interactive Compounds Clement Lin and Danzhou Yang 18 Analysis of Telomere-Homologous DNA with Different Conformations Using 2D Agarose Electrophoresis and In-Gel Hybridization Zepeng Zhang, Qian Hu, and Yong Zhao 19 Analysis of Telomere Proteins by Chromatin Immunoprecipitation (ChIP) Feng Liu, Xuyang Feng, and Wenbin Ma 139 147 161 171 197 205 Index 215 Contributors Eladio Abreu  •  Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA, USA; Department of Genetics, University of Georgia, Athens, GA, USA Susan M. Bailey  •  Department of Environmental & Radiological Health Sciences, Colorado State University, Fort Collins, CO, USA Christopher J. Bley  •  Department of Chemistry and Biochemistry, Arizona State University, Tempe, AZ, USA Sandy Chang  •  Department of Laboratory Medicine, Yale University School of Medicine, New Haven, CT, USA Julian J.L. Chen  •  Department of Chemistry and Biochemistry, Arizona State University, Tempe, AZ, USA Michael N. Cornforth  •  Department of Radiation Oncology, University of Texas Medical Branch, Galveston, TX, USA Xuyang Feng  •  Key Laboratory of Gene Engineering of the Ministry of Education, State Key Laboratory for Biocontrol, Department of Biochemistry, School of Life Sciences, Sun Yat-sen University, Guangzhou, China Edwin H. Goodwin  •  KromaTiD Inc., Fort Collins, CO, USA Min Hsu  •  Department of Microbiology & Immunology, W.R. Hearst Microbiology Research Center, Weill Medical College of Cornell University, New York, NY, USA Qian Hu  •  Key Laboratory of Gene Engineering of the Ministry of Education, Higher Education Mega Center, School of Life Sciences, Sun Yat-sen University, Guangzhou, China Jing Huang  •  State Key laboratory of Molecular Biology, National Center for Protein Science Shanghai, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of, Chinese Academy of Sciences, Shanghai Science Research Center, Chinese Academy of Sciences, Shanghai, China Junjiu Huang  •  Key Laboratory of Gene Engineering of the Ministry of Education, SYSU-BCM Joint Center for Biomedical Sciences and Institute of Healthy Aging Research, School of Life Sciences, Sun Yat-sen University, Guangzhou, China Shuai Jiang  •  Key Laboratory of Gene Engineering of the Ministry of Education, SYSU-BCM Joint Center for Biomedical Sciences and Institute of Healthy Aging Research, School of Life Sciences, Sun Yat-sen University, Guangzhou, China Hyeung Kim  •  Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX, USA Li Lan  •  University of Pittsburgh Cancer Institute, Pittsburgh, PA, USA; Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA Ok-Hee Lee  •  Department of Biomedical Science, CHA University, Seongnam-si, Gyeonggi-do, Republic of Korea; Severance Integrative Research Institute for Cerebral and Cardiovascular Diseases, Yonsei University Health System, Seoul, Republic of Korea Ming Lei  •  State Key laboratory of Molecular Biology, National Center for Protein Science Shanghai, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese ix x Contributors Academy of Sciences, Shanghai, China; Shanghai Science Research Center, Chinese Academy of Sciences, Shanghai, China Clement Lin  •  Medicinal Chemistry and Molecular Pharmacology, College of Pharmacy, Purdue University, West Lafayette, IN, USA Dan Liu  •  Cell-Based Assay Screening Service Core, Baylor College of Medicine, Houston, TX, USA; Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX, USA Feng Liu  •  Key Laboratory of Gene Engineering of the Ministry of Education, State Key Laboratory for Biocontrol, Department of Biochemistry, School of Life Sciences, Sun Yat-sen University, Guangzhou, China Arturo Londoño-Vallejo  •  Telomeres & Cancer Laboratory, CNRS-UMR3244, Institut Curie, Paris, France; UPMC University Paris 06, Paris, France Neal F. Lue  •  Department of Microbiology & Immunology, W.R. Hearst Microbiology Research Center, Weill Medical College of Cornell University, New York, NY, USA Wenbin Ma  •  Key Laboratory of Gene Engineering of the Ministry of Education, State Key Laboratory for Biocontrol, Department of Biochemistry, School of Life Sciences, Sun Yat-sen University, Guangzhou, People’s Republic of China Miles J. McKenna  •  Department of Environmental & Radiological Health Sciences, Colorado State University, Fort Collins, CO, USA; KromaTiD Inc., Fort Collins, CO, USA Asha S. Multani  •  Department of Laboratory Medicine, Yale University School of Medicine, New Haven, CT, USA Isabelle Ourliac-Garnier  •  Telomeres & Cancer Laboratory, CNRS-UMR3244, Institut Curie, Paris, France; UPMC University Paris 06, Paris, France Rekha Rai  •  Department of Laboratory Medicine, Yale University School of Medicine, New Haven, CT, USA Dustin P. Rand  •  Department of Chemistry and Biochemistry, Arizona State University, Tempe, AZ, USA Erin Robinson  •  KromaTiD Inc., Fort Collins, CO, USA Jerry W. Shay  •  Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX, USA Zhou Songyang  •  Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX, USA Hidetoshi Tahara  •  Department of Cellular and Molecular Biology, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan Mengfan Tang  •  Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Michael P. Terns  •  Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA, USA; Department of Genetics, University of Georgia, Athens, GA, USA Rebecca M. Terns  •  Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA, USA; Department of Genetics, University of Georgia, Athens, GA, USA Rong Tan  •  University of Pittsburgh Cancer Institute, Pittsburgh, PA, USA; Xiangya Hospital, Central South University, Changsha, Hunan, China; University of Pittsburgh School of Medicine, Pittsburgh, PA, USA Woodring E. Wright  •  Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX, USA Contributors xi Huawei Xin  •  Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA Jun Xu  •  Cell-Based Assay Screening Service Core, Baylor College of Medicine, Houston, TX, USA Danzhou Yang  •  Medicinal Chemistry and Molecular Pharmacology, College of Pharmacy, Purdue University, West Lafayette, IN, USA; Purdue Center for Cancer Research, Purdue University, West Lafayette, IN, USA; Purdue Institute for Drug Discovery, Purdue University, West Lafayette, IN, USA Zepeng Zhang  •  Key Laboratory of Gene Engineering of the Ministry of Education, Higher Education Mega Center, School of Life Sciences, Sun Yat-sen University, Guangzhou, China Yong Zhao  •  Key Laboratory of Gene Engineering of the Ministry of Education, Higher Education Mega Center, School of Life Sciences, Sun Yat-sen University, Guangzhou, China 204 Zepeng Zhang et al recombination-dependent telomere maintenance Mol Cell 42:224–236 Henson JD, Cao Y, Huschtscha LI, Chang AC, Au AY, Pickett HA, Reddel RR (2009) DNA C-circles are specific and quantifiable markers of alternative-lengthening-of-telomeres activity Nat Biotechnol 27:1181–1185 Cesare AJ, Griffith JD (2004) Telomeric DNA in ALT cells is characterized by free telomeric circles and heterogeneous t-loops Mol Cell Biol 24:9948–9957 Nabetani A, Ishikawa F (2009) Unusual telomeric DNAs in human telomerase-negative immortalized cells Mol Cell Biol 29:703–713 10 Cohen S, Agmon N, Yacobi K, Mislovati M, Segal D (2005) Evidence for rolling circle replication of tandem genes in Drosophila Nucleic Acids Res 33:4519–4526 11 Cohen S, Segal D (2009) Extrachromosomal circular DNA in eukaryotes: possible involvement in the plasticity of tandem repeats Cytogenet Genome Res 124:327–338 12 Bell L, Byers B (1983) Separation of branched from linear DNA by two-dimensional gel electrophoresis Anal Biochem 130:527–535 Chapter 19 Analysis of Telomere Proteins by Chromatin Immunoprecipitation (ChIP) Feng Liu, Xuyang Feng, and Wenbin Ma Abstract Telomere chromatin immunoprecipitation (ChIP) is an experimental method used to determine whether proteins are associated with telomere DNA inside the nuclei of cells and tissues Telomere-associated proteins are first covalently crosslinked to telomere DNA, and then immunoprecipitated using an antibody specific for the protein of interest This method has become one of the most indispensable tools for investigating the protein complexes that associate with telomeres Key words Telomere chromatin immunoprecipitation, ChIP, Telomere-associated proteins 1  Introduction Telomeres are protected by a multitude of proteins, several of which exhibit specific binding activity for telomere DNA sequences Among the telomere DNA-binding proteins identified, TRF1 and TRF2 associate with double-stranded telomere DNA [1], whereas POT1 binds single-stranded telomere overhangs [2] These proteins in turn associate with other signaling proteins to achieve telomere-­ end protection and length control [3–5] Telomere targeting can be assessed in a variety of assays, and telomere chromatin immunoprecipitation (ChIP) has emerged as an indispensable tool for examining the architecture of telomere chromatin and elucidating how different players localize to telomeres and interact with other telomere recruited factors [6] Here, a nonradioactive method is described to analyze immunoprecipitated protein-DNA complexes 2  Materials 2.1  Cell Preparation 10× Phosphate buffered saline (PBS): Dissolve g KCl, 80 g NaCl, 17.8 g Na2HPO4·2H2O, and 2.4 g KH2PO4 in 800 mL distilled water, and adjust pH to 7.4 with HCl Make final Zhou Songyang (ed.), Telomeres and Telomerase: Methods and Protocols, Methods in Molecular Biology, vol 1587, DOI 10.1007/978-1-4939-6892-3_19, © Springer Science+Business Media LLC 2017 205 206 Feng Liu et al volume to 1 L with distilled water Autoclave and store at room temperature 37% formaldehyde solution A working solution of 1% (v/v) is freshly prepared in 1× PBS or medium not supplemented with serum Stop solution: Prepare 2 M Glycine stock solution, filter through 0.22 μm filter, and store at room temperature Final working concentration is 0.125 M Complete protease inhibitors (Roche Molecular Biochemicals) Solution A: 10 mM Hepes, pH 7.9, mM EDTA, 0.5 mM EGTA, 0.25% Triton X-100, and freshly added complete protease inhibitors Solution B: 10 mM Hepes, pH 7.9, mM EDTA, 0.5 mM EGTA, 200 mM NaCl, and freshly added complete protease inhibitors Solution C: 0.1% sodium deoxycholate, 150 mM NaCl, 50 mM Hepes, pH 7.5, 0.1% SDS, 1% Triton X-100, mM PMSF, and freshly added complete protease inhibitors 20 mg/mL Proteinase K solution Sonicator (such as the VIRSONIC 600, The Virtis Company) 10 Cell scrapers (for adherent cells) 11 Tube Shaker/Rotator 12 20% SDS (w/v) 2.2  Preclearing E coli DNA preparation Dissolve E coli DNA (Sigma D-2001, type VIII) in 10 mM Tris–HCl (pH 7.5) at a final concentration of 2.5 mg/mL. Sonicate the DNA solution to obtain a fragment pool of ~500 bp (see Subheading 3.1) Purify the fragmented DNA once by phenol/chloroform/isoamylalcohol (25:24:1) extraction and once by chloroform extraction Collect the aqueous phase and add 1/10 volume of 3 M sodium acetate solution Mix well and add two volumes absolute ethanol Centrifuge the mix at top speed to pellet the DNA. Wash the pellet once with 70% ethanol Resuspend E coli DNA in 1× TE and measure DNA concentration at OD 260 Make mg/ mL E coli DNA working stock and store at −20 °C 5% BSA (w/v) Dissolve g of Bovine serum albumin Fraction V in 100 mL of 1× PBS. Sterilize through a 0.22 μm filter and store at °C Prepare preclearing protein A/G-agarose bead slurry the day before immunoprecipitation Mix μg sheared E coli DNA and 10 μL 5% BSA with 50 μL protein A-agarose beads in a microcentrifuge tube Shake/mix the solution overnight at °C for use the next day Telomere ChIP 2.3  Immuno-­ precipitations 207 Control immunoglobulins such as IgG 1 M dithiothreitol (DTT) Dissolve 1.54 g of DTT in H2O plus 33.33 μL of 3 M NaOAc (pH 5.2) to make a final volume of 10 mL. Sterile filter the solution, aliquot, and freeze at −20 °C. Working concentration is mM ChIP I buffer: 0.1% sodium deoxycholate, 150 mM NaCl, 1% Triton X-100, mM EDTA, 50 mM Hepes, pH 7.5 Add complete protease inhibitors and DTT right before use ChIP II buffer: 0.1% sodium deoxycholate, 500 mM NaCl, 1% Triton X-100, mM EDTA, 50 mM Hepes, pH 7.5 Add complete protease inhibitors and DTT right before use ChIP III buffer: 0.5% sodium deoxycholate, 0.25 M LiCl, 0.5% NP-40, mM EDTA, 10 mM Tris–HCl, pH 8.0 Add complete protease inhibitors and DTT right before use TE/DTT buffer: 10 mM Tris–HCl, pH 8.0, mM EDTA, pH 8.0, and mM DTT. Add DTT right before use Elution Buffer: 0.5% SDS, 0.1 M NaHCO3 Make fresh from 10% SDS and 1 M NaHCO3 before use 5 M NaCl 2.4  Telomere DNA Purification 0.5 M EDTA, pH 8.0 1 M Tris–HCl, pH 6.5 DNase-free RNase A, 10 mg/mL QIAquick PCR purification kit (Qiagen) 2.5  Slot Blot Hybond-N+ membrane (GE Healthcare) and 3 M Whatman filter paper Bio-Dot SF microfiltration apparatus (Bio-Rad) 20× SSC: Dissolve 175.3 g NaCl and 88.2 g sodium citrate in ddH2O to a final volume of 1 L. Adjust pH to 7.0 with 10 N NaOH, autoclave, and store at room temperature Denaturing Solution: 1.5 M NaCl, 0.5 M NaOH Neutralizing Solution: 3 M NaCl, 0.5 M Tris–HCl, pH 7.0 A UV crosslinker Biotin 3′ end DNA labeling Kit (Pierce) T4 Polynucleotide Kinase (NEB) 1 M sodium phosphate buffer, pH 7.2: 134 g Na2HPO4•7H2O in 1 L ddH2O. Stir until dissolved Adjust pH to 7.2 with H3PO4, and store at room temperature Chemiluminescent 10 North2South® Detection Kit (Pierce) Hybridization and 208 Feng Liu et al 11 Hybridization buffer: Mix 250 mL of 1 M sodium phosphate buffer (pH 7.2), mL of 0.5 mM EDTA (pH 8.0), 175 mL of 20% SDS, and ddH2O to a final volume of 500 mL. Filter-­ sterilize and store at −20 °C. Warm to 55 °C to dissolve any precipitates before use 3  Methods 3.1  Cell Lysis and Chromatin Shearing Grow cells to confluence (on three 15 cm dishes) (see Note 1) Replace medium with 10–15 mL of freshly prepared 1% formaldehyde working solution Rock the plates gently for 10 min at room temperature (see Note 2) Remove the crosslinking solution, and wash the cells twice with ice-cold 1× PBS Add 10 mL of 1× PBS, and 625 μL of 2 M Glycine (to a final concentration 0.125 M) to each dish Mix gently and incubate at room temperature for 15 min to quench the formaldehyde and stop the crosslinking reaction Wash the cells twice with 20 mL of ice-cold 1× PBS. Aspirate off the PBS after each wash Carefully scrape cells into a 50 mL conical tube with a cell scraper Pellet cells at 1,000 g for 5 min at °C and discard the supernatant Add mL of Solution A to the pellet, vortex, and centrifuge in a microcentrifuge at 4,000 rpm (1,500 g) for 5 min at °C, discard the supernatant Repeat step with mL of Solution B 10 Add mL of Solution C. Vortex to mix well 11 Sonicate the samples on ice until the lysate clears Sonication should be carried out in bursts with intervals of cooling on ice In general, cycles of 10 s sonication (at power setting 4) followed by 30 s of cooling on ice will suffice The lysate should remain ice-cold throughout the sonication step (see Note 3) 12 Transfer the sonicated samples to fresh prechilled 1.5 mL microcentrifuge tubes Centrifuge at 14,000 rpm (18,400 g) for 15 min at °C. Transfer the supernatant, which contains sheared chromatin, to fresh prechilled 1.5 mL microcentrifuge tubes The samples can be used directly or flash frozen in liquid N2 and stored at −80 °C 3.2  Preclearing and Immunoprecipitation For every 900 μL of sonicated lysate, add 50 μL of the preclearing protein A/G-agarose bead slurry from Subheading 2.2 and μg of control immunoglobulin (e.g., IgG) If frozen Telomere ChIP 209 samples are used, thaw the lysate on ice and then centrifuge at 14,000 rpm (18,400 g) for 15 min at °C. Transfer the supernatant to a fresh 1.5 mL microcentrifuge tube (see Note 4) Incubate at °C with rocking (or on a rotator) for 2–4 h Centrifuge the sample at 3,000 rpm (845 g) for 5 min at °C Transfer the supernatant to a new microcentrifuge tube Remove ~40 μL and save as the input sample Divide the remaining lysate equally for each IP (~400 μL), and add μg of the desired antibody or appropriate IgG, together with 25 μg of sheared E coli DNA plus μL of 5% BSA. Rock or rotate the samples at °C for h to overnight (see Note 5) Prepare fresh protein A/G beads just before use as follows Aliquot appropriate amount of the bead slurry into a clean prechilled microcentrifuge tube Add 0.5–1 mL of Solution C Spin down the beads at 3,000 rpm (845 g) for 1 min at °C Resuspend with Solution C to ~50% slurry For each IP, 20 μL of 50% slurry is needed Add 20 μL of the slurry to each IP tube Rotate for another 1–2 h at °C Centrifuge at 3,000 rpm (845 g) for 1 min at °C. Gently aspirate the supernatant Add 400 μL of ChIP I buffer Rotate for 10 min at °C Repeat step once with ChIP II Repeat step once with ChIP III Repeat step once with TE/DTT 10 Centrifuge at 3,000 rpm (845 g) for 1 min at °C. Discard the supernatant 3.3  Elution and DNA Purification Add 100 μL of freshly prepared Elution buffer to each sample Incubate for 10 min at room temperature, vortex every 2 min Centrifuge at 3,000 rpm (845 g) for 1 min at room ­temperature, and transfer the supernatant to a new microcentrifuge tube Steps and may be repeated one to two times to increase total DNA recovered Combine the eluate Add 1/25 (v/v) of 5 M NaCl Mix and incubate overnight at 65 °C Briefly spin the tubes For every 300 μL of eluted DNA sample, add μL of 0.5 M EDTA, 12 μL of 1 M Tris-HCl (pH 6.5), and 1.2 μL of DNase-free RNase A (from 10 mg/mL stock) Incubate at 37 °C for 30 min To every 300 μL of eluted DNA sample, add μL of Proteinase K (from 20 mg/mL stock) Incubate at 37 °C for h 210 Feng Liu et al Purify the DNA using the QIAquick PCR purification kit Mix the sample with the PB buffer supplied with the kit and agitate for 30 min before loading the sample onto the spin column Elute DNA in 50~100 μL of the elution buffer supplied with the kit (see Note 6) 3.4  Slot Blot Moisten Hybond-N+ membrane (12 × cm, mark edge for orientation) with ddH2O, and then soak it in 2× SSC for 10 min (see Note 7) Clean, dry, and assemble the Bio-Dot Microfiltration Apparatus according to the instruction manual Load 100 μL of 2× SSC and switch on the vacuum until all liquid drains from the wells Switch the vacuum off until ready to load samples Boil the DNA samples for min, cool on ice, and spin briefly in a microfuge to collect all samples Load 200 μL of 2× SSC in each well Then load the desired amount of DNA samples (30–100 μL) Mix well with the 2× SSC (see Note 8) Switch on the vacuum to gently remove buffer Disassemble Bio-Dot SF microfiltration apparatus Place the membrane on a piece of 3 M Whatman paper saturated with denaturing solution for 10 min with the DNA side up Place the membrane on another piece of 3 M Whatman paper saturated with Neutralizing solution for 10 min with the DNA side up Gently blot the membrane on dry filter paper to remove excess liquid Crosslink the DNA to the membrane immediately in a UV crosslinker (120 mJ/cm2) Rinse in 2× SSC. Allow the membrane to air dry 10 Pre-hybridize the membrane with 10 mL of hybridization buffer in a sealed plastic bag or roller bottle for h at 45 °C 11 Prepare and optimize the working concentration of biotin-­ labeled (TTAGGG)3 probe (see Note 9) (a) A nonradioactive 5′- biotinylated (TTAGGG)3 probe may be obtained commercially As an alternative, the (TTAGGG)3 probe can be 3′- labeled and purified with the Biotin 3′ end DNA labeling Kit (Pierce) following the manufacturer’s instruction Specifically, mix in a microcentrifuge tube μL of the oligonucleotide probe (1 μM), 10 μL of 5× Terminal Deoxynucleotidyl Transferase (TdT) buffer, μL of Biotin-­11-­UTP (5 μM), μL of TdT (1.5 U/μL), and 25 μL of ultrapure water Incubate the reaction at 37 °C for 30 min Add 2.5 μL EDTA (0.2 M) to the tube to stop the reaction Add 50 μL of phenol/ Telomere ChIP 211 chloroform/isoamyl alcohol (25:24:1) to each reaction Vortex the mixture and centrifuge 2 min at 12,000 rpm (13,500 g) to separate the different phases Carefully remove the top aqueous phase for later (b)  Make a serial dilution of the biotinylated (TTAGGG)3 probe (e.g., 100, 50, 20, 10, 5, 2, and nM) to estimate the best working concentration If the labeling efficiency is more than 70%, 10 nM of the biotinylated (TTAGGG)3 probe usually generates good results 12 Discard pre-hybridization solution Add 10 mL of fresh hybridization buffer together with 10 μL of the labeled probe (1:1000) and hybridize overnight at 45 °C 13 Wash the membrane once at room temperature and once at 37 °C in 4× SSC, 0.1% SDS for 10 min each 14 Wash the membrane in 2× SSC, 0.1% SDS for 10 min at 45 °C (see Note 10) 15 Wash the membrane in 2× SSC for 5 min at room temperature 16 Block the membrane by adding 20 mL of Blocking Buffer from the North2South® Chemiluminescent Hybridization and Detection Kit and incubate at room temperature for 15 min with gentle shaking 17 Replace the Block Buffer with the conjugate solution from North2South® Chemiluminescent Hybridization and Detection Kit (1:300 streptavidin-HRP) and incubate at room temperature for 15 min with gentle shaking 18 Wash the membrane at room temperature in Wash Buffer four times for 5 min each with gentle shaking 19 Transfer the membrane to Substrate Equilibration Buffer and shake gently for 5 min at room temperature 20 Remove excess buffer and add Luminol/Enhancer Solution from North2South® Chemiluminescent Hybridization and Detection Kit onto the membrane Incubate for 5 min without shaking 21 Wrap the membrane with plastic wrap and expose the membrane to X-ray films or an appropriate CCD camera Quantify the signals and calculate the fold of enrichment (Fig. 1) 4  Notes Different cell types will yield different numbers of cells For cells such as Hela, each confluent 15 cm dish yields 1–2 × 107 cells, enough for IPs, whereas one 15 cm dish of primary cells may be enough for IP. A 0.5 mL cell suspension (1 × 107 Feng Liu et al anti-TAH1 IgG * 2.5 2.0 1.5 1.0 0.5 an tiT AH Ig G 10% input Percentage of input (%) e pr ob Al u Te lo m er e pr ob e 212 Fig Telomere ChIP analysis using U2OS cells and the biotinylated telomere probe (TTAGGG)3 Anti-TAH1 antibodies were used to precipitate endogenous TAH1 and the telomere DNA associated with it IgG served as a negative control An Alu probe was used to control for total amount of DNA loaded Left, blotting of the membrane with the probes Right, quantification of the data Error bars indicate standard error (n = 3) *p < 0.05, calculated by the Student t-test cells/mL) is desirable for each IP experiment However, we have obtained excellent results using far fewer cells when obtaining large numbers of cells is impossible The duration of crosslinking and the concentration of formaldehyde can be adjusted according to the protein of interest The extent of crosslinking is critical, too much crosslinking may mask epitopes while too little may result in incomplete fixation DNA should be sheared to an average fragment size of ~500– 1,000 bp Optimal sonication conditions are important for ChIP efficiency Sonication conditions differ for each cell type, fixation protocol, and sonicator apparatus To check the sonication result, mix 10 μL of the sonicated sample with μL of Protease K (20 mg/mL) in a microcentrifuge tube and incubate at 37 °C for 20 min Add drop of phenol, vortex, and centrifuge at 14,000 rpm for 1 min Use the supernatant to run on DNA agarose gels to determine fragment size The control immunoglobulin should ideally be species and isotype matched to the primary antibody of interest It is also advisable to save a small aliquot of the lysate for western blot analysis It is important to include all necessary controls including controls for primary antibodies and positive controls for the experiment Plan ahead so there is enough material for all of the samples Telomere ChIP 213 Alternatively, DNA can be also purified using the following method (a) Add 24 μg of Proteinase K, incubate for h at 37 °C (b) Cool the samples to room temperature, add 400 μL of phenol/chloroform/isoamyl alcohol (25:24:1), vigorously vortex for s (c) Centrifuge for 2 min at 13,000 rpm (15,800 g) at RT. Transfer the aqueous phase to a microcentrifuge tube (d) Add 12 μg of glycogen (from 20 mg/mL stock) and 2.5× volumes of ice-cold 100% ethanol, mix well, and precipitate the DNA overnight at −20 °C (e) Centrifuge the samples at 14,000 rpm (18,400 g) for 20 min at °C (f) Carefully remove the supernatant and add 500 μL of icecold 70% ethanol to the pellet (g) Centrifuge the samples at 14,000 rpm (18,400 g) for 20 min at °C. Carefully remove the supernatant, air dry the pellet until all traces of ethanol have evaporated (>30 min) (h) Resuspend pellet in 100 μL of ddH2O or 10 mM Tris– HCl (pH 7.5) The DNA is now ready for downstream applications or being aliquoted and stored at −80 °C Always use forceps or wear gloves when handling membranes and 3 M paper Mixing 2× SSC with DNA samples ensures even distribution of the sample on the membrane The apparatus has different vacuum settings When loading the DNA sample, vacuuming should be on the gentle setting Nonradioactive biotin-labeled (TTAGGG)3 probe can be replaced with a radiolabeled (TTAGGG)3 probe (a) Prepare radiolabeled (TTAGGG)3 probe by mixing 10 pmol of oligonucleotide, μL of T4PNK buffer (10×), μL of 32P-γ-ATP (3,000 Ci/mmol), μL of T4 Polynucleotide Kinase, and μL of ddH2O. Incubate at 37 °C for h Add 80 μL of TE (pH 8.0) and incubate for 5 min at 70 °C to stop the reaction (b) Purify the probe using QIAquick nucleotide removal kit (QIAGEN) Alternatively, the probe can also be purified by adding 30 μL of 10 M ammonium acetate, μL of 10 mg/mL tRNA, and 400 μL ethanol Mix by vortex, centrifuge the tube at 14,000 rpm (18,400 g) for 10 min at °C. Wash the pellet with 70% ethanol, and then dissolve the pellet in 100 μL TE (pH 8.0) Check the radioactivity on a liquid scintillation counter Specific activity should be around 0.5–1 × 106 cpm/μL 214 Feng Liu et al (c) Discard pre-hybridization solution Add 10 mL fresh hybridization buffer together with the labeled probe (≥1 × 106 cpm/mL) and hybridize overnight at 50 °C (d) Wash the membrane in 4× SSC, 0.1% SDS for 10 min once at room temperature, and once at 37 °C (e) Wash the membrane in 2× SSC, 0.1% SDS for 10 min at 50 °C. The blot should be carefully monitored using a Geiger counter throughout the wash The membrane should be ready for exposure once the edges or areas without samples no longer set off the Geiger counter The number or temperature of washes can be adjusted accordingly (f) Wash the membrane in 2× SSC for 5 min at room temperature (g) Wrap the membrane with plastic wrap for autoradiography Expose either to films (10–24 h) or in a phosphoImager cassette (1.5–3 h) 10 Usually, the membrane will need to be stripped and hybridized to a control probe (e.g., the Alu probe) It is critical that the membrane should stay moist throughout the experiment Acknowledgment  The authors would like to thank Drs Jiancong Liang and Dong Yang for their support and suggestions This work was supported by the National Natural Science Foundation of China (NSFC 31570827 and 31501108), and the Science and Technology Planning Project of Guangdong Province (2015B020228002) References Broccoli D, Smogorzewska A, Chong L, de Lange T (1997) Human telomeres contain two distinct Myb-related proteins, TRF1 and TRF2 Nat Genet 17:231–235 Baumann P, Cech TR (2001) Pot1, the putative telomere end-binding protein in fission yeast and humans Science 292: 1171–1175 de Lange T (2005) Shelterin: the protein complex that shapes and safeguards human telomeres Genes Dev 19:2100–2110 Liu D, O’Connor MS, Qin J, Songyang Z (2004) Telosome, a mammalian telomere-­ associated complex formed by multiple telomeric proteins J Biol Chem 279:51338–51342 Songyang Z, Liu D (2006) Inside the mammalian telomere interactome: regulation and regulatory activities of telomeres Crit Rev Eukaryot Gene Expr 16:103–118 de Lange, T (2010) Chromatin Immuno precipitation (ChIP) http://delangelab.rockefeller edu/protocols_files/ChIP_protocol.pdf Index A ACX primer Affinity��������������������31, 42, 43, 51, 84, 86, 157, 187, 189–191 Affinity tags������������������������������������������������������������������������84 Aging��������������������������������������������3, 4, 30, 105, 140, 161, 172 Amino-allyl (C6 amino-modified dT) DNA probes�������115 Antibody�����������������21, 25, 85, 89, 92, 114–121, 123, 137, 138, 141, 144, 145, 149, 151, 155, 172, 173, 209, 212 Anticancer drugs Aplastic anemia��������������������������������������������������������������������4 Attrition����������������������������������������������������������������������� 4, 133 B Bi-molecular fluorescence complementation (BiFC)������������������������������������ 147–152, 156–158 BrdU incorporation 5-Bromo-2′-deoxycytidine (BrdC)����������� 45, 52, 105, 107, 109 5′-Bromo-2′-deoxyuridine (BrdU) BrdU labeling������������������������������������������������������� 75, 123 C Cajal body��������������������������������������������������114, 117–119, 122 Cancers�����������������������������������3, 4, 15, 36, 49, 71, 81, 95, 114, 116–118, 122, 140, 162, 171–173, 175, 197 Cell cycle G1/S�������������������������������������������������� 72, 75, 81, 120, 121 S-phase��������������42, 43, 49, 72, 75, 81, 107, 114, 118–121 synchronization���������������������������������������������� 73, 75, 120 Cell lysis���������������������������������������������������������������������������208 Cell synchronization����������������������������������������������������� 75, 81 Chromatin immunoprecipitation (ChIP)����������������� 212, 213 Chromosomal fusion centromere–centromere (C–C) fusion������������������������130 centromere–telomere (C–T) fusion����������������������������130 end-to-end fusion����������������������������������������� 2–4, 55, 197 telomere–telomere (T–T) fusion���������������������������������130 Chromosomes��������������������������������������������������������������������42 stability�������������������������������������������������������������������������65 Compounds�������������������������������������������������66, 117, 148, 193 Coplin jars����������������������������������������� 106, 108, 109, 111, 137 C-rich fill-in�����������������������������������������������������������������������71–81 PNA telomeric probe�������������������� 31, 34, 44, 47, 75, 105 CRISPR/Cas9 guide RNA (gRNA)��������������������������������6, 16–21, 23–25 knockout cell line�����������������������������������������15, 18, 20, 21 protospacer adjacent motif (PAM)������������������������� 16, 24 Streptococcus pyogenes Cas9 (SpCas9)������������������������������ 16 Crystallization�������������������������������������������161–168, 173, 179 CsCl gradients�������������������������������������������������� 72, 74–77, 81 Cytogenetic analysis�������������������������������������������������127–131 D DEAE chromatography�����������������������������������������������������87 Dehydration����������������������������������������������163, 167, 168, 172 Denaturation�����������������������������������17, 22, 51, 107, 110, 121, 123, 124, 199, 203 Density�������������������������������������72, 76–81, 151, 153, 156, 158 DNA double-strand breaks (DSBs)��������������6, 104, 127, 133 Domains������������������������������������������������ 2, 147, 149, 158, 162 Duplex specific nuclease (DSN)�������56, 57, 62, 72, 74, 78, 79 Dysfunction����������������������������������3–6, 30, 131, 133, 134, 140 E Escherichia coli��������������������������������������������� 57, 115, 118, 153, 162–164, 199, 206, 209 EGTA������������������������������������������������������� 66, 84, 96, 98, 206 Electron microscopy�����������������������������������������������������������56 Electropheresis alkaline agarose gels������������������������������������������ 57, 59–60 DNA ladder�������������������������������������������������17, 22, 26, 27 TBE buffer��������������������������������������������������� 26, 200, 201 Electroporation������������������������������������������������������� 17, 19, 24 Elution���������������������������������26, 162, 164, 200, 207, 209–210 Extracts��������������������������������������������������16, 17, 19, 20, 22, 25, 26, 79, 80, 84–87, 89–91, 96, 151, 176, 178, 191, 206 F Fixation������������������������������������������32, 45, 117, 121, 124, 212 Flow cytometer�����������������������������24, 121, 148, 150, 152, 157 Fluorescence in situ hybridization (FISH) CO-FISH�������������������������������� 6, 42–47, 50–52, 104, 105 NextGen CO-FISH���������������������������������������������������111 Q-FISH����������������������������������������������������������� 43, 51–53 Telo-dGH™����������������������������������������������������������������111 Zhou Songyang (ed.), Telomeres and Telomerase: Methods and Protocols, Methods in Molecular Biology, vol 1587, DOI 10.1007/978-1-4939-6892-3, © Springer Science+Business Media LLC 2017 215 Telomeres and Telomerase: Methods and Protocols 216  Index    Fluorescence microscopy coverslip������������������������115, 117, 118, 120, 122–124, 134 dye�������������������������������������������������������115–118, 122, 123 Fluorescent protein (FP) YFP������������������������������������������������������������ 148–150, 156 Insect cell Interactome�������������������������������������������������������� 2–3, 147 Intramolecular���������������������������������������������������������� 172, 173 In vitro telomerase activity�����������������������������������������������162 In vitro transcription and translation��������������������������������162 G K Genomic DNA DSN digestion�������������������������������������������������� 57–59, 79 extraction�����������������������������������������������������19, 20, 22, 25 restriction digestion������������������������������������������������ 16, 17 sample preparation��������������������������������������������������������22 yield������������������������������������������������������������������������������25 Giemsa staining�������������������������������������������������������� 128, 129 Glycosidic bonds��������������������������������������������������������������171 G-overhang length�����������������������������������������������������55–62, 65–70, 72 G-quadruplex folding��������������������������172, 178, 180–182, 185, 186, 189 G-tetrad����������������������������������������������171–173, 175, 176, 179–183, 185, 187 intramolecular��������������������������������������172–176, 179, 180 ligand������������������������������������������� 172, 173, 175–176, 186 structure������������������������������������������������������ 140, 171–193 telomeric����������������������������������������������������� 174–175, 193 topology�����������������������������������������������180–182, 189, 193 G-quadruplex-interactive ligands�������������������������������������175 G-rich LNA telomeric probe��������������������������������� 43, 44, 47 G-tail telomere������������������������������������������������������� 65, 68, 69 GTU4������������������������������������������������������������57, 60, 199, 201 Guanines��������������������������� 171–175, 179–184, 186, 187, 192 Killerred��������������������������������������������������������������������139–145 Knockout (KO)����������������������������������� 4–6, 15, 18, 20, 21, 91 KR-TEL system���������������������������������������������������������������140 H Hela cell�����������������25, 73, 75, 81, 96, 99, 116, 117, 119–122, 135, 136, 140–142, 144, 150, 157, 176, 211 Hexameric��������������������������������������������������������������������������71 Hexamers���������������������������������������������������������������������������55 High-throughput���������������������������������������������������������� 66, 96 Hoogsteen hydrogen bonding�������������������������� 171, 172, 179 Hybridization����������������������������������������35, 48, 51, 61, 74, 77, 79, 107, 198, 201, 202 non-denaturing�������������������������������������������������������������55 probe C-rich 51, 61, 74, 77, 79, 198, 201, 202 telomere-specific��������������������������������35, 48, 107, 198 Hybridization protection assay (HPA)��������������55, 65, 68, 69 I IgG-sepharose pull down��������������������������������������������� 87, 91 Image analysis���������������������������������������������32, 34–35, 47–48 Image capture��������������������������������������������������32, 44, 58, 199 Immunoprecipitation������������������������������������������������205–214 Indels������������������������������������������������������������������������������ 6, 24 L Lagging lagging daughter����������������������������������������������� 72, 74–78 lagging strand��������������������������������������42, 51, 77–78, 104 Lagging overhangs������������������������������������������������� 72, 78–80 Leading leading daughter����������������������������������������������������� 72, 78 leading strand������������������������������������������42, 51, 104, 105 Ligands������������������������������������������������������������� 173, 175, 176 Light activation������������������������������������������������ 142–143, 145 LNA�������������������������������������������������31, 37, 44, 46, 47, 50, 51 Luciferase Gaussia luciferase (Gluc)���������������������148–150, 152–153, 157, 158 M Malignancy��������������������������������������������������������������������� 4, 95 Medaka fish����������������������������������������������������������������������162 Metaphase chromosomes��������������������������������34, 42, 47, 106 Metaphase spreads�������������������������������������� 31, 33, 36, 42, 44, 45, 50, 108, 109 Metaphase suspensions�������������������������� 31, 32, 36, 43, 45, 50 Mouse embryonic fibroblast (MEF)����������������� 128, 134, 136 N NMR spectroscopy guanine base resonance���������������������������������������180–181 spectral assignment������������������������������������� 180, 182–185 thymine/cytosine base resonances�������������������������������181 Noncanonical��������������������������������������������������������������������171 Nonhomologous end joining (NHEJ)������������������ 6, 104, 127 O Oxidative stress�����������������������������������������������������������������140 8-Oxoguanine (8-oxoG)���������������������������������� 140, 144, 145 P Peptide array������������������������������������������������������������� 149, 159 Peptide nucleic acid (PNA)�����������������������123, 128, 135, 137 Peptide synthesis��������������������������������������������������������������158 Permeablize��������������������������������117, 124, 135, 136, 141, 143 Telomeres and Telomerase: Methods and Protocols 217 Index       PNA������������������������31, 33, 36, 37, 43, 44, 46–48, 50, 51, 130 PNA telomere FISH������������������������������������������������128–130 Preclearing�������������������������������������������������������� 206, 208–209 Primer�������������������������������������������������2, 19, 21, 56, 62, 84, 96 Primer extension assays������������������������������������������ 84, 88, 91 Primers������������������������������������������������������������������������� 17, 23 Protease HRV 3C����������������������������������������������������� 163, 164 Protein complementation assay (PCA)����������������������� 6, 147–159 expression����������������������������������������������������������� 155, 164 GST-fusion protein������������������������������������ 153–155, 159 recombinant protein�������������������������������������������162–163 Protein purification��������������������������������������������������164–165 Proteinase K������������������������������������������ 25, 85, 206, 209, 213 Protein–protein interaction������������2, 3, 6, 147, 149–153, 159 R Radiolabeled telomeric probe���������������������������������������������23 Replication������������������������������������2–4, 29, 41–43, 51, 53, 65, 71, 72, 75, 104, 113, 121, 140, 172, 173 Retrovirus infection��������������������������������������������������������������134–136 production���������������������������������������������������������� 134, 136 titer�����������������������������������������������������������������������������135 Revesz syndrome������������������������������������������������������������������4 Ribonucleoprotein (RNP)�������������������������� 65, 71, 83, 86, 88, 95, 127, 161, 162 S SDS-PAGE����������������������������������85, 151, 155, 158, 164, 165 Senescence�������������������������������������������������������������3, 4, 30, 71 Single-stranded DNA�������������������������������107, 175, 178, 179 Slot blot����������������������������� 74, 75, 77–81, 207–208, 210–211 Southern blot hybridization����������������������������������������������������������������56 probe�����������������������������������������������������������������������������56 random label�����������������������������������������������������������������26 Stem cells����������������������������������������������������������36, 50, 71, 95 Structure polymorphism���������������������������������������������������175 Structures��������������������������������������1–3, 5, 15, 35, 49, 55, 58, 65, 66, 75, 114, 118, 122, 127, 131, 133, 140, 149, 161, 162, 166, 193, 198 Synchronization�����������������������������������������������73, 75, 81, 120 T 293T cells����������������������� 19, 25, 134–136, 138, 150, 156, 157 T4 DNA ligase������������������������������������������������������������� 17, 19 T7 endonuclease I�������������������������������������������������� 17, 20, 24 Telomerase������������������������������������������������������������������������162 activity������������������������������3, 29, 31, 43, 65, 66, 86, 87, 91, 95–100, 113, 114, 122, 162, 173, 175, 176 processivity������������������������������������������������������� 84, 89–90 substrate������������������������������������������������������������������ 55, 96 telomerase reverse transcriptase (TERT) 2–4, 83, 86, 113, 161, 162 TRBD domain�����������������������������������������������������162 telomerase RNA������������������������������� 3, 83, 85–87, 89–91, 113, 116, 119, 122 TERC(TR) CR4/5 domain������������������������������������������������������162 TRAP assay�������������������������������������������������84, 95, 96, 99 Telomerase activity������������������������������������������������������� 65, 66 Telomerase inhibitors������������������������������������������������� 95, 162 Telomerase protein component������������������������������ 85–86, 89 Telomerase visualization����������������������������������� 114–120, 122 fluorescence in situ hybridization hTR FISH�������������������������������������������� 114–120, 122 immunofluorescence��������������������114–116, 118–121, 134 Telomere D-loop������������������������������������������������������������������ 2, 3, 55 double-stranded DNA�����������������������������56, 72, 127, 203 human cells��������������������������� 3, 6, 15, 56, 65, 72, 136, 197 recombination���������������������������������������� 4, 29, 30, 42, 197 shortening�������������������������������������������� 4, 30, 41, 127, 140 single-stranded DNA�������������������� 79, 107, 175, 178, 179 subtelomeric�������������������������������������������������������������������1 telomeric DNA������������������������������ 1, 2, 57, 59, 62, 65, 75, 111, 122, 123, 140, 161, 172–175, 179, 180, 197 telomeric repeat���������������������������������2, 16, 29, 41, 57, 84, 100, 133, 199 T-loop�����������������������������������������������������������2, 55, 65, 66 Telomere binding proteins POT1������������������������� 2–5, 42, 65, 66, 122, 127, 133, 205 RAP1������������������������������������������2, 3, 5, 65, 122, 133, 134 TIN2 TPP1��������������������������������������������� 2–5, 65, 127, 133, 135 TRF1���������2–4, 42, 65, 119, 122, 127, 133, 140–144, 205 TRF2�����������������������������������2–4, 18–21, 65, 66, 118, 122, 127, 133–135, 147, 205 Telomere damage response������������������������������� 133–138, 140 Telomere dysfunction diseases�����������������������������������������������������������������������3–5 genome stability����������������������������������������������������������3–5 Telomere induced foci (TIF)���������������������������������������� 6, 135 Telomere interactome post-translational modifications (PTM)������������������������3 Telomere length�����������������������������������5, 6, 15–27, 41, 51, 66, 67, 71, 113, 140, 161, 197 Telomere maintenance������������������������1, 3, 5, 16, 83, 113, 140 Telomere replication����������������������������������������������� 41–43, 72 Telomere restriction fragment (TRF) assay������������������ 16, 72 Telomere-induced foci (TIF)�������������������������������������������134 Telomeric repeat amplification protocol (TRAP)������������������������������������������������ 84, 95, 99 cell proliferation������������������������������������������������������ 30, 41 malignancy PCR������������������������������������������������������������������ 84, 99 Telomeres and Telomerase: Methods and Protocols 218  Index    Telomeric repeat amplification protocol (TRAP) (cont.) senescence���������������������������������������������������������������95 telomerase���������������������������������������������������������������95 Telomeric-oligonucleotide ligation assay (T-OLA)�����������56 Thymidine���������������������������������������49, 72, 73, 75, 77–79, 81, 104, 116, 120, 121 T-loop������������������������������������������������������������������������������133 T4 polynucleotide kinase������������������������� 17, 18, 26, 207, 213 Transcription in vitro�������������������������������������������������������� 163, 165–167 Transfection��������������������������������� 17, 19, 21, 23, 24, 136, 141, 142, 144, 150–152, 156, 157 TS primer U U2OS cells������������� 96, 99, 135, 136, 140–142, 144, 150, 212 V Vector������������������������������������������16, 18–21, 23, 24, 107, 136, 149, 150, 155, 156, 158, 163 W Western blotting�������������������������������� 21, 25, 78, 87, 157, 212 Werner syndrome (WRN)����������������������������������������� 3, 4, 42 Y Yeast telomerase�����������������������������������������������������������83–92 ... possession an ever-expanding arsenal that continues to aid us in dissecting the function of telomerase and telomere-binding proteins, probing the changes in telomeres, and elucidating the consequences... (ed.), Telomeres and Telomerase: Methods and Protocols, Methods in Molecular Biology, vol 1587, DOI 10.1007/978-1-4939-6892-3_1, © Springer Science+Business Media LLC 2017 Zhou Songyang and complexity... changes in turn impact the length of telomeres and the status of the cell such as its replicative Zhou Songyang (ed.), Telomeres and Telomerase: Methods and Protocols, Methods in Molecular Biology,

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Mục lục

  • Preface

  • Contents

  • Contributors

  • Chapter 1: Introduction to Telomeres and Telomerase

    • 1 Telomere Structure

    • 2 The Telomere Interactome: An Integrated Telomere Signaling Network

    • 3 Telomere Dysfunction, Genome Stability, and Diseases

    • 4 Tools for Studying Telomere Biology

    • References

    • Chapter 2: Analysis of Average Telomere Length in Human Telomeric Protein Knockout Cells Generated by CRISPR/Cas9

      • 1 Introduction

      • 2 Materials

        • 2.1 For the Generation of KO Cells by CRISPR/Cas9

        • 2.2 For TRF Analysis

        • 3 Methods

          • 3.1 Using CRISPR/Cas9 to Generate Telomeric Protein KO Cells

            • 3.1.1 CRISPR Vector Construction

            • 3.1.2 Validate CRISPR/Cas9 gRNA Vector Cleavage Efficiency (See Note 4.1.8)

            • 3.1.3 Generation and Validation of CRISPR/Cas9 KO Cells

            • 3.2 TRF Analysis Using the CRISPR KO Cells

              • 3.2.1 Sample Preparation

              • 3.2.2 Electrophoresis and Transfer

              • 3.2.3 Hybridization and Analysis

              • 4 Notes

                • 4.1 CRISPR/Cas9 KO Cells

                • 4.2 TRF Analysis

                • References

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