Growth Conditions for SMC Proteins v1
Authors: L. Marston Adele , Robertson Daniel , Makrantoni Vasso 
Published: September 2021

A plethora of biological processes like gene transcription, DNA replication, DNA recombination, and chromosome segregation are mediated through protein–DNA interactions. A powerful method for investigating proteins within a native chromatin environment in the cell is chromatin immunoprecipitation (ChIP). Combined with the recent technological advancement in next generation sequencing, the ChIP assay can map the exact binding sites of a protein of interest across the entire genome. Here we describe a-step-by step protocol for ChIP followed by library preparation for ChIP-seq from yeast cells. Chromatin immunoprecipitation (ChIP) is a powerful method for assaying protein–DNA binding in vivo and is broadly used to estimate the density of DNA-bound proteins at specific sites in the genome. ChIP is a multistep assay and every step needs to be optimized for consistent results. Briefly, protein–DNA associations are immobilized by cross-linking with formaldehyde [1,2,3] before shearing the chromatin, either mechanically [4] or by enzymatic digestion [5] into DNA fragments of average size 200–500bp. Specific cross-linked protein–DNA complexes are then isolated by immunoprecipitation using an antibody to the protein of interest. Finally, the cross-links are reversed, and the retrieved DNA is analyzed to determine the sequences bound by the protein. ChIP followed by quantitative real-time PCR (ChIP-qPCR), using specific primers, can be used to measure protein association and relative abundance at a particular genomic locus. Alternatively, ChIP can be combined with next generation sequencing (ChIP-seq) to provide a genome-wide view of protein occupancy. While ChIP-seq allows for relative protein abundance at distinct chromosomal addresses to be compared within a sample, differences between samples cannot be quantified without introducing a method to normalize. Typically, this involves “spike in” of a known amount of DNA or cross-linked cells from a different species, with sufficient sequence divergence from the organism of interest to allow sequencing reads to be confidently distinguished bioinformatically [6,7,8]. This technique, referred to as calibrated ChIP-seq, makes it possible to quantitate genome-wide changes in the distribution of an epitope tagged protein and allows for quantification of differences in occupancy between experimental samples [8]. Calibrated ChIP-seq requires that both calibration and experimental organisms carry the same epitope tag and can be immunoprecipitated by the same protocol. For this protocol we use S. pombe to calibrate S. cerevisiae, a combination that also allows us to invert the roles, that is, calibrate S. pombe with S. cerevisiae. The ChIP method described here has been optimized for use with chromatin from two species of yeast,S. cerevisiae and S. pombe; however, it should be easy to adapt it for use with other chromatin sources. To demonstrate the robustness of our ChIP and library preparation protocols we performed ChIP against the Scc1 subunit of the cohesin multiprotein complex, tagged with the 6HA epitope [9,10,11] . We have also used a similar protocol for the condensin subunit Brn1 [12] and for the meiotic counterpart of cohesin, Rec8 [13]. Here, we outline in detail an optimized protocol for cross-linking and harvesting cells, fragmenting chromatin, immunoprecipitating the desired protein–DNA complexes, and preparing the library for sequencing on the Illumina MiniSeq platform. A schematic stepwise representation of the method is illustrated in Fig.1. References: Solomon MJ, Varshavsky A (1985) Formaldehyde-mediated DNA-protein crosslinking: a probe for in vivo chromatin structures. Proc Natl Acad Sci U S A 82(19):6470–6474 Solomon MJ, Larsen PL, Varshavsky A (1988) Mapping protein-DNA interactions in vivo with formaldehyde: evidence that histone H4 is retained on a highly transcribed gene. Cell 53(6):937–947 Gilmour DS, Lis JT (1984) Detecting protein-DNA interactions in vivo: distribution of RNA polymerase on specific bacterial genes. Proc Natl Acad Sci U S A 81(14):4275–4279 Kuo MH, Allis CD (1999) In vivo cross-linking and immunoprecipitation for studying dynamic protein:DNA associations in a chromatin environment. Methods 19(3):425–433.https://doi.org/10.1006/meth.1999.0879 Thorne AW, Myers FA, Hebbes TR (2004) Native chromatin immunoprecipitation. Methods Mol Biol 287:21–44.https://doi.org/10.1385/1-59259-828-5:021 Bonhoure N, Bounova G, Bernasconi D, Praz V, Lammers F, Canella D, Willis IM, Herr W, Hernandez N, Delorenzi M, Cycli XC (2014) Quantifying ChIP-seq data: a spiking method providing an internal reference for sample-to-sample normalization. Genome Res 24(7):1157–1168.https://doi.org/10.1101/gr.168260.113 Orlando DA, Chen MW, Brown VE, Solanki S, Choi YJ, Olson ER, Fritz CC, Bradner JE, Guenther MG (2014) Quantitative ChIP-Seq normalization reveals global modulation of the epigenome. Cell Rep 9(3):1163–1170.https://doi.org/10.1016/j.celrep.2014.10.018 Hu B, Petela N, Kurze A, Chan KL, Chapard C, Nasmyth K (2015) Biological chromodynamics: a general method for measuring protein occupancy across the genome by calibrating ChIP-seq. Nucleic Acids Res 43(20):e132.https://doi.org/10.1093/nar/gkv670 Fernius J, Marston AL (2009) Establishment of cohesion at the pericentromere by the Ctf19 kinetochore subcomplex and the replication fork-associated factor, Csm3. PLoS Genet 5(9):e1000629.https://doi.org/10.1371/journal.pgen.1000629 Fernius J, Nerusheva OO, Galander S, Alves Fde L, Rappsilber J, Marston AL (2013) Cohesin-dependent association of scc2/4 with the centromere initiates pericentromeric cohesion establishment. Curr Biol 23(7):599–606.https://doi.org/10.1016/j.cub.2013.02.022 Hinshaw SM, Makrantoni V, Kerr A, Marston AL, Harrison SC (2015) Structural evidence for Scc4-dependent localization of cohesin loading. elife 4:e06057.https://doi.org/10.7554/eLife.06057 Verzijlbergen KF, Nerusheva OO, Kelly D, Kerr A, Clift D, de Lima Alves F, Rappsilber J, Marston AL (2014) Shugoshin biases chromosomes for biorientation through condensin recruitment to the pericentromere. elife 3:e01374.https://doi.org/10.7554/eLife.01374 Vincenten N, Kuhl LM, Lam I, Oke A, Kerr AR, Hochwagen A, Fung J, Keeney S, Vader G, Marston AL (2015) The kinetochore prevents centromere-proximal crossover recombination during meiosis. elife 4.https://doi.org/10.7554/eLife.10850 Nelson JD, Denisenko O, Bomsztyk K (2006) Protocol for the fast chromatin immunoprecipitation (ChIP) method. Nat Protoc 1(1):179–185.https://doi.org/10.1038/nprot.2006.27 Cockram CA, Filatenkova M, Danos V, El Karoui M, Leach DR (2015) Quantitative genomic analysis of RecA protein binding during DNA double-strand break repair reveals RecBCD action in vivo. PNAS Aug 25;112(34):E4735–42. DeAngelis MM, Wang DG, Hawkins TL (1995) Solid-phase reversible immobilization for the isolation of PCR products. Nucleic Acids Res 23(22):4742–4743 Ewels P, Magnusson M, Lundin S, Kaller M (2016) MultiQC: summarize analysis results for multiple tools and samples in a single report. Bioinformatics 32(19):3047–3048.https://doi.org/10.1093/bioinformatics/btw354 Li H (2018) Minimap2: pairwise alignment for nucleotide sequences. Bioinformatics 34(18):3094–3100.https://doi.org/10.1093/bioinformatics/bty191 Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R, 1000 Genome Project Data Processing Subgroup (2009) The sequence alignment/map format and SAMtools. Bioinformatics 25(16):2078–2079.https://doi.org/10.1093/bioinformatics/btp352 Quinlan AR, Hall IM (2010) BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26(6):841–842.https://doi.org/10.1093/bioinformatics/btq033 Robinson JT, Thorvaldsdottir H, Winckler W, Guttman M, Lander ES, Getz G, Mesirov JP (2011) Integrative genomics viewer. Nat Biotechnol 29(1):24–26.https://doi.org/10.1038/nbt.1754 Tian B, Yang J, Brasier AR (2012) Two-step cross-linking for analysis of protein-chromatin interactions. Methods Mol Biol 809:105–120.https://doi.org/10.1007/978-1-61779-376-9_7 Craig DW, Pearson JV, Szelinger S, Sekar A, Redman M, Corneveaux JJ, Pawlowski TL, Laub T, Nunn G, Stephan DA, Homer N, Huentelman MJ (2008) Identification of genetic variants using bar-coded multiplexed sequencing. Nat Methods 5(10):887–893.https://doi.org/10.1038/nmeth.1251 Ford E, Nikopoulou C, Kokkalis A, Thanos D (2014) A method for generating highly multiplexed ChIP-seq libraries. BMC Res Notes 7:312. https://doi.org/10.1186/1756-0500-7-312 Acknowledgements: We are grateful to Manu Shukla for discussions and comments on the ChIP-seq library preparation and for kindly providing a representative Bioanalyzer image, Nicholas Toda, Jesus Torres-Garcia, and Flora Paldi for sharing tips on the ChIP-seq library protocol and Stefan Galangher and Lesley Clayton for general comments on the manuscript. This work was funded by Wellcome through a Senior Research Fellowship to AM and a Wellcome Centre core grant [107827 and 203149].