TITLE: Growth Conditions for SMC Proteins
AUTHORS: Adele L. Marston, Daniel Robertson, Vasso Makrantoni

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">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.</div><br/><div class = "text-block"><span>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 [</span><a href="https://link.springer.com/protocol/10.1007/978-1-4939-9520-2_10#CR1" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:UNDERLINE;">1</span></a><span style = ":;:UNDERLINE;:UNDERLINE;:UNDERLINE;">,</span><a href="https://link.springer.com/protocol/10.1007/978-1-4939-9520-2_10#CR2" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":UNDERLINE;:UNDERLINE;">2</span></a><span style = ":UNDERLINE;:UNDERLINE;:UNDERLINE;font-style:italic;">,</span><a href="https://link.springer.com/protocol/10.1007/978-1-4939-9520-2_10#CR3" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":UNDERLINE;font-style:italic;">3</span></a><span>] before shearing the chromatin, either mechanically [</span><a href="https://link.springer.com/protocol/10.1007/978-1-4939-9520-2_10#CR4" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;font-style:italic;">4</span></a><span>] or by enzymatic digestion [</span><a href="https://link.springer.com/protocol/10.1007/978-1-4939-9520-2_10#CR5" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":UNDERLINE;font-style:italic;">5</span></a><span>] into DNA fragments of average size 200–500 bp. 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 [</span><a href="https://link.springer.com/protocol/10.1007/978-1-4939-9520-2_10#CR6" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:UNDERLINE;">6</span></a><span style = ":;:;:UNDERLINE;:UNDERLINE;">,</span><a href="https://link.springer.com/protocol/10.1007/978-1-4939-9520-2_10#CR7" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:UNDERLINE;">7</span></a><span style = ":;:;:UNDERLINE;:UNDERLINE;">,</span><a href="https://link.springer.com/protocol/10.1007/978-1-4939-9520-2_10#CR8" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:UNDERLINE;">8</span></a><span>]. 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 [</span><a href="https://link.springer.com/protocol/10.1007/978-1-4939-9520-2_10#CR8" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:UNDERLINE;">8</span></a><span>]. 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 </span><span style = ":;">S. pombe </span><span>to calibrate </span><span style = ":;">S. cerevisiae</span><span>, a combination that also allows us to invert the roles, that is, calibrate </span><span style = ":;">S. pombe </span><span>with </span><span style = "font-style:italic;">S. cerevisiae</span><span>.</span></div><div class = "text-block"><span>The ChIP method described here has been optimized for use with chromatin from two species of yeast,</span><span style = "font-style:italic;">S. cerevisiae </span><span>and </span><span style = "font-style:italic;">S. pombe</span><span>; 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 [</span><a href="https://link.springer.com/protocol/10.1007/978-1-4939-9520-2_10#CR9" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:UNDERLINE;">9</span></a><span style = ":;:;:UNDERLINE;:UNDERLINE;">,</span><a href="https://link.springer.com/protocol/10.1007/978-1-4939-9520-2_10#CR10" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:UNDERLINE;">10</span></a><span style = ":;:;:UNDERLINE;:UNDERLINE;">,</span><a href="https://link.springer.com/protocol/10.1007/978-1-4939-9520-2_10#CR11" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:UNDERLINE;">11</span></a><span>] . We have also used a similar protocol for the condensin subunit Brn1 [</span><a href="https://link.springer.com/protocol/10.1007/978-1-4939-9520-2_10#CR12" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:UNDERLINE;">12</span></a><span>] and for the meiotic counterpart of cohesin, Rec8 [</span><a href="https://link.springer.com/protocol/10.1007/978-1-4939-9520-2_10#CR13" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:UNDERLINE;">13</span></a><span>]. 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.</span><a href="https://link.springer.com/protocol/10.1007/978-1-4939-9520-2_10#Fig1" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:UNDERLINE;">1</span></a><span>.</span></div><br/><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/files/ee4h9nan.jpg" /></div><br/><div class = "text-block"><span style = "font-weight:bold;">References:</span></div><div class = "text-block"><ol style = "list-style-type: decimal;"><li style = "counter-reset:ol0;">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</li><li style = "counter-reset:ol0;">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. 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BMC Res Notes 7:312. </span><a href="https://doi.org/10.1186/1756-0500-7-312" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://doi.org/10.1186/1756-0500-7-312</span></a></li></ol></div><br/><div class = "text-block"><span style = "font-weight:bold;">Acknowledgements:</span></div><div class = "text-block">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].</div><br/></div>

[STEPS]
?. [Growth Conditions for SMC Proteins]
For depletion of Cdc20, use a construct where CDC20 is under control of the methionine-repressible promoter, pMET3 (pMET3-CDC20).
S. cerevisiae strains for mitotic studies are grown in YPDA at  . The most consistent results, at least for cohesin, are obtained when cells are arrested in metaphase of mitosis prior to the ChIP procedure. This can be achieved either by depletion of the anaphase-promoting complex subunit, Cdc20, or treatment of the cells with the microtubule-depolymerizing drug nocodazole.
?. [Growth Conditions for SMC Proteins]
Briefly for Cdc20 depletion, dilute an overnight culture to OD600 = 0.2 in minimal media lacking methionine and grow for – at  to  OD600 = 0.3–0.4.
25 °C
?. [Growth Conditions for SMC Proteins]
Dilute culture back to OD600 = 0.2 in same media and arrest cells in G1 by adding  for  and  for an additional .
[α-factor]
[α-factor]
?. [Growth Conditions for SMC Proteins]
Check microscopically that at least 90% of cells are arrested before collecting on a filter (Whatman ME25, 0.45 μm), and wash with 10 volumes of medium lacking sugar with the aid of a vacuum pump.
?. [Growth Conditions for SMC Proteins]
Quickly resuspend cells in YPDA containing  and re-add methionine to  every .
[methionine]
?. [Growth Conditions for SMC Proteins]
Harvest cells after – in a metaphase arrest confirmed by microscopy.
?. [Growth Conditions for SMC Proteins]
For nocodazole arrest, follow these subsequent steps:
?. [Growth Conditions for SMC Proteins]
Dilute an overnight culture to OD600 = 0.2 in YPDA and grow for – at  to OD600 = 0.3–0.4.
25 °C
?. [Growth Conditions for SMC Proteins]
Dilute culture back to OD600 = 0.2 in YPDA media containing a mixture of  and .
[nocodazole ]
[benomyl ]
?. [Growth Conditions for SMC Proteins]
Read nocodazole every  at  . Harvest cells after – confirming metaphase arrest by microscopy.
?. [Growth Conditions for SMC Proteins]
For inducing meiosis, follow these subsequent steps:
For studies of protein occupancy during meiosis we use diploid S. cerevisiae strains of SK1 background including (a)REC8-3HA ndt80Δ (AM4015), as previously described [13] and (b)REC8-6HIS-3FLAG (AM11000).
?. [Growth Conditions for SMC Proteins]
Recover SK1 strains from  on YPG agar plates  at , before transferring to YPDA4% agar plates for a further - at .
-80 °C
30 °C
30 °C
?. [Growth Conditions for SMC Proteins]
Inoculate cultures in liquid YPDA at  with shaking for ~, prior to inoculating into BYTA medium to OD600 = 0.3 .
30 °C
?. [Growth Conditions for SMC Proteins]
The next morning, spin cells down, wash with dH2O and resuspend in SPO medium to OD600 = 1.8 and shake at .
30 °C
?. [Growth Conditions for SMC Proteins]
For prophase I arrest (ndt80Δ) for Rec8 cells, harvest  after resuspension in sporulation medium and confirm arrest by FACS.
[media]
S. pombe strains used for calibration are listed in the materials under "Yeast Strains and Growth Material" and are grown in YES at  .