Cross-Linking and Cell Harvesting

Vasso Makrantoni, Daniel Robertson, Adele L. Marston

Published: 2021-09-22 DOI: 10.17504/protocols.io.bn3hmgj6

Abstract

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.

Before start

For ChIP-qPCR:* For cohesin subunit, Scc1 , in cycling cells, harvest 50 ml yeast cells of density 0.3–0.6 OD₆₀₀grown in YPDA media.
Note
Alternatively, cells can be arrested in mitosis either by treatment with nocodazole or by depletion of Cdc20, as described in protocol "Growth Conditions for SMC Proteins".
* For the less abundant cohesin loader subunit Scc2 and condensin subunit Brn1 harvest 100 ml yeast cells of density 0.3–0.6 OD₆₀₀grown in YPDA.

For the meiotic counterpart of cohesin, Rec8 , harvest 50 ml yeast cells of density 1.8 OD₆₀₀grown in SPO media.

For ChIP-seq:* Grow 2× the amount of cell culture of yeast cells of density 0.3–0.6 OD₆₀₀in YPDA media (100 ml Scc1 , 200 ml Scc2 and Brn1 ) or 1.8 OD₆₀₀in SPO media (100 ml Rec1 ) and process each 50 ml sample individually.
Note
For calibrated ChIP-seq use a 2:1 ratio of S. cerevisiae to S. pombe cells, as measured by OD600, mix pellets of different organisms in a single fastprep tube and lyse together as previously described [For calibrated ChIP-seq use a 2:1 ratio of S. cerevisiae to S. pombe cells, as measured by OD600, mix pellets of different organisms in a single fastprep tube and lyse together as previously described [8]. Use the same batch of S. pombe in all samples of the same experiment. Perform each IP individually and pool samples together after the final wash step by combining beads from multiple IPs in the same 200 μl of TES elution buffer (see Step 4 in protocol "Immunoprecipitation, Decross-linking, and DNA Extraction"). Both calibration and experimental genomes need to express proteins tagged with the same epitope for immunoprecipitation and the calibration organism needs to be sufficiently similar that the ChIP protocol works for both.]. Use the same batch of S. pombe in all samples of the same experiment. Perform each IP individually and pool samples together after the final wash step by combining beads from multiple IPs in the same 200 μl of TES elution buffer (see Step 4 in protocol "Immunoprecipitation, Decross-linking, and DNA Extraction"). Both calibration and experimental genomes need to express proteins tagged with the same epitope for immunoprecipitation and the calibration organism needs to be sufficiently similar that the ChIP protocol works for both.

Steps

Cross-Linking and Cell Harvesting

Cross-link cells by adding 5mL to give a final concentration of 1% formaldehyde in the culture.

Gently rotate on an orbital shaker at 90rpm for Scc1, Rec8, Scc2, and Brn1.

Note

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 [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].].

To quench cross-linking, add glycine at a final concentration of 125millimolar (mM).

Note

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.

Incubate with gentle shaking for 0h 5m 0s atRoom temperature.

Collect cells by centrifugation at 1800x g,4°C,0h 0m 0s.

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:

6.1.

Wash cells in 10mL. (1/2)

6.2.

Wash cells in 10mL. (2/2)

6.3.

Wash with 10mL.

Collect cells by centrifugation at 1800x g,4°C,0h 0m 0s.

Carefully aspirate the supernatant and snap freeze pellets in liquid nitrogen in fastprep screw-cap tubes. Store the pellets at -80°C until ready to use.

Cross-Linking and Cell Harvesting

Abstract

Before start

Steps

Cross-Linking and Cell Harvesting

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