TITLE: Cell Lysis and Sonication
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]
?. [Cell Lysis and Sonication]
Thaw cells . Add 1 volume (–) supplemented with 0.5% SDS,  and protease inhibitors (Roche Complete EDTA-free tablet).
on ice
0.3 mL
[ice-cold 1× FA lysis buffer]
[PMSF]
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.
?. [Cell Lysis and Sonication]
Add an equal volume (–) and lyse cells in a FastPrep-24 Homogenizer at  for  (homogenization speed 6.5). Leave  for .
200 µl
[0.5-mm Zirconium Silicate beads]
4 °C
on ice
?. [Cell Lysis and Sonication]
Repeat this twice more: Add an equal volume (–) and lyse cells in a FastPrep-24 Homogenizer at  for  (homogenization speed 6.5). Leave  for .(1/2)
200 µl
[0.5-mm Zirconium Silicate beads]
4 °C
on ice
?. [Cell Lysis and Sonication]
Add an equal volume (–) and lyse cells in a FastPrep-24 Homogenizer at  for  (homogenization speed 6.5). Leave  for .(2/2)
200 µl
[0.5-mm Zirconium Silicate beads]
4 °C
on ice
?. [Cell Lysis and Sonication]
Place samples .
on ice
?. [Cell Lysis and Sonication]
Dry the outside of the tubes, invert and puncture the tube bottom with a red flamed (under a Bunsen burner) 25G × 5/8″ needle.
?. [Cell Lysis and Sonication]
Immediately place the fastprep tube within a chilled 15 ml conical Falcon tube already containing an empty fastprep tube and centrifuge at .
Centrifuge: 1250 34, 3 min, 4 10
?. [Cell Lysis and Sonication]
Transfer the entire lysate (both pellet and supernatant) to a prechilled 1.5 ml Eppendorf tube. Centrifuge at .
Centrifuge: 16000 34, 15 min, 4 10
?. [Cell Lysis and Sonication]
Remove the supernatant by vacuum aspiration and resuspend the pellets thoroughly in  supplemented with 0.1% SDS,  and protease inhibitors.
[ice-cold 1× FA buffer]
[PMSF]
?. [Cell Lysis and Sonication]
Centrifuge at . Discard supernatant.
Centrifuge: 16000 34, 15 min, 4 10
Notice the presence of a pellet with a glass-like layer. This is the cross-linked chromatin.
?. [Cell Lysis and Sonication]
Resuspend washed chromatin pellets well in  containing 0.1% SDS,   and protease inhibitors.
[ice-cold 1× FA buffer]
[PMSF]
?. [Cell Lysis and Sonication]
Shear the chromatin by sonication, using a Bioruptor Twin with circulating water bath at a temperature of less than  and power settings: High, /, 30 cycles.
5 °C
[ON]
[OFF]
Sonication conditions must be determined empirically for each cell type, and sonicator model; the optimal average DNA fragment size is below 0.5 kb. Overfragmentation of chromatin is not recommended as it can damage the protein epitope targeted by the antibody of choice. If the Diagenode water bath sonicator is not available, a probe sonicator or Covaris instrument can also be used. Sonication time and intensity will need to be optimized and DNA fragment size determined as in protocol "Determine the Size of Sonicated Samples and the DNA Concentration".
?. [Cell Lysis and Sonication]
Centrifuge the sonicated mixture at  to remove cell debris and transfer the supernatant into a new cold Eppendorf tube containing  with 0.1% SDS, , protease inhibitors.
Centrifuge: 16000 34, 15 min, 4 10
[1× FA lysis buffer]
[PMSF]
?. [Cell Lysis and Sonication]
Mix by inversion and centrifuge at .
Centrifuge: 16000 34, 15 min, 4 10
?. [Cell Lysis and Sonication]
Carefully transfer the supernatant into a new, cold Eppendorf tube. This chromatin preparation will be used for the immunoprecipitation in protocol "Immunoprecipitation, Decross-linking, and DNA Extraction".
The amount of SDS in immunoprecipitation can interfere with antibody binding efficiency therefore, lower amount of SDS can also be used. Either omit SDS in 2xFA buffer or reduce SDS added afterward to a final concentration of 0.05% before chromatin immunoprecipitation.
?. [Cell Lysis and Sonication]
Store  at . This will be the “Input” sample.
[supernatant]
-20 °C
?. [Cell Lysis and Sonication]
Use  (from step 13) to determine fragment size as determined in protocol "Determine the Size of Sonicated Samples and the DNA Concentration".
[chromatin preparation]