Cell Lysis and Sonication

Vasso Makrantoni, Daniel Robertson, Adele L. Marston

Published: 2021-09-22 DOI: 10.17504/protocols.io.bn3pmgmn

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

Steps

Cell Lysis and Sonication

Thaw cells On ice. Add 1 volume (0.3mL–0.5mL) supplemented with 0.5% SDS, 1millimolar (mM) and protease inhibitors (Roche Complete EDTA-free tablet).

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.

Add an equal volume (200µL–250µL) and lyse cells in a FastPrep-24 Homogenizer at 4°C for 0h 0m 30s (homogenization speed 6.5). Leave 4On ice for 0h 10m 0s.

2.1.

Repeat this twice more: Add an equal volume (200µL–250µL) and lyse cells in a FastPrep-24 Homogenizer at 4°C for 0h 0m 30s (homogenization speed 6.5). Leave 4On ice for 0h 10m 0s.

(1/2)

2.2.

Add an equal volume (200µL–250µL) and lyse cells in a FastPrep-24 Homogenizer at 4°C for 0h 0m 30s (homogenization speed 6.5). Leave 4On ice for 0h 10m 0s.

(2/2)

Place samples 4On ice.

Dry the outside of the tubes, invert and puncture the tube bottom with a red flamed (under a Bunsen burner) 25G × 5/8″ needle.

Immediately place the fastprep tube within a chilled 15 ml conical Falcon tube already containing an empty fastprep tube and centrifuge at 1250x g,4°C.

Transfer the entire lysate (both pellet and supernatant) to a prechilled 1.5 ml Eppendorf tube.

Centrifuge at 16000x g,4°C.

Remove the supernatant by vacuum aspiration and resuspend the pellets thoroughly in 1mL supplemented with 0.1% SDS, 1millimolar (mM) and protease inhibitors.

Centrifuge at 16000x g,4°C. Discard supernatant.

Note

Notice the presence of a pellet with a glass-like layer. This is the cross-linked chromatin.

Resuspend washed chromatin pellets well in 0.3mL containing 0.1% SDS, 1millimolar (mM) and protease inhibitors.

10.

Shear the chromatin by sonication, using a Bioruptor Twin with circulating water bath at a temperature of less than 5°C and power settings: High, 0h 0m 30s/0h 0m 30s, 30 cycles.

Note

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".

11.

Centrifuge the sonicated mixture at 16000x g,4°C to remove cell debris and transfer the supernatant into a new cold Eppendorf tube containing 1mL with 0.1% SDS, 1millimolar (mM), protease inhibitors.

12.

Mix by inversion and centrifuge at 16000x g,4°C.

13.

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".

Note

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.

14.

Store 10µL at -20°C. This will be the “Input” sample.

15.

Use 100µL (from step 13) to determine fragment size as determined in protocol "Determine the Size of Sonicated Samples and the DNA Concentration".

Cell Lysis and Sonication

Abstract

Before start

Steps

Cell Lysis and Sonication

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