TITLE: Cross-Linking and Cell Harvesting
AUTHORS: Vasso Makrantoni, Daniel Robertson, Adele L. Marston

[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></div>

[STEPS]
?. [Cross-Linking and Cell Harvesting]
Cross-link cells by adding  to give a final concentration of 1% formaldehyde in the culture.
[11% formaldehyde in diluent buffer]
?. [Cross-Linking and Cell Harvesting]
Gently rotate on an orbital shaker at  for Scc1, Rec8, Scc2, and Brn1.
33
The cross-linking time and formaldehyde concentration can affect both the efficiency of chromatin shearing and of antigen precipitation. Shorter cross-linking times (5–10 min), lower formaldehyde concentrations (1%, wt/vol), or both, may improve shearing efficiency. However, for some proteins, especially those that do not directly bind DNA, this might reduce the efficiency of cross-linking and thus the yield of precipitated chromatin. It is advisable to perform a cross-linking time course to determine optimal fixation conditions.In vivo cross-linking for ChIP is traditionally achieved with formaldehyde; however, formaldehyde is a short spacer arm cross-linker (2 Å), limiting its functionality. For higher order interactions, longer cross-linkers such as EGS (16.1 Å) or DSG (7.7 Å) or combination of cross-linkers can be used so to more efficiently trap larger protein complexes with complex quaternary structure [22].
?. [Cross-Linking and Cell Harvesting]
To quench cross-linking, add glycine at a final concentration of .
Addition of glycine to quench the formaldehyde is particularly important when harvesting large volumes of cell culture as the harvesting process can be long and thereby can increase fixation time between samples.
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Incubate with gentle shaking for  at.
0 Room temperature
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Collect cells by centrifugation at .
Centrifuge: 1800 34, 4 10
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Wash cells twice in 10 ml ice-cold TBS buffer and once in 10 ml ice-cold 1× FA lysis buffer supplemented with 0.1% SDS:
?. [Cross-Linking and Cell Harvesting]
Wash cells in . (1/2)
[ice-cold TBS buffer]
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Collect cells by centrifugation at .
Centrifuge: 1800 34, 4 10
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Carefully aspirate the supernatant and snap freeze pellets in liquid nitrogen in fastprep screw-cap tubes. Store the pellets at  until ready to use.
-80 °C
?. [Cross-Linking and Cell Harvesting]
Wash cells in . (2/2)
[ice-cold TBS buffer]
?. [Cross-Linking and Cell Harvesting]
Wash with .
[ice-cold 1× FA lysis buffer supplemented with 0.1% SDS]