GSM4024111: Lsh-/- NPC_H3K27me3_ChIPSeq; Mus musculus; ChIP-Seq
For each sample 9x10^6 cells were harvested by trypsinisation and rinsed well in 1xPBS before being resuspended in 1.5ml of PBS. Each sample was fixed with the addition of 40.5 µl of 37% formaldehyde (Sigma) and incubated for 10minutes at room temperature on rotation. 225 µl of 1M glycine was added to quench the formaldehyde and rotated at room temperature for 5minutes. Samples were then centrifuged at 2000rpm for 4 minutes at 4 oC, supernatant discarded and pellets resuspended in 2ml LB1 (50mM Hepes KOH pH7.9, 140mM NaCl,1mM EDTA, 10% glycerol, 0.5% IPEGAL, 0.25% triton X100, 1xComplete EDTA-free protease inhibitor tablet (Roche)) solution and rotated at 4 oC for 4 minutes. This process was repeated resuspending in 2 ml LB2 (10mM TrisHcl pH8, 200mM NaCl, 1mM EDTA, 0.5mM EGTA, 1xComplete EDTA-free protease inhibitor tablet (Roche)) solution and then repeated resuspending in 200 µl LB3 (10mM TrisHCl Ph8, 100mM NaCl, 1mM EDTA, 0.5mM EGTA, 0.1% Na Deoxycholate, 0.5% LaurylSulphate, 1xComplete EDTA-free protease inhibitor tablet (Roche)). Chromatin was then sheared at 4 oC using a Diagenode bioruptor set on high for 45 cycles at 30seconds on/30seconds off. 20 µl of 10% triton X in LB3 was then added to each sample and mixed by inversion. Samples were then centrifuged at 13000rpm at 4 oC for 10 minutes to remove nuclear debris and supernatant transferred to a fresh tube. In order to check the efficiency of the sonication process, 8 µl of sheared chromatin was taken, mixed with 1 µl 5M NaCl and 16 µl dH2O, incubated at 98 oC for 15minutes and treated with RNase cocktail before being purified with Qiagen PCR Clean up kit as per manufacturer's instructions . Sheared chromatin was then resolved on 1.5% agarose gel post-stained with EtBr. The DNA smear should be between 100-500bp with peak at 250-300bp. For immunoprecipitation 25 µl of Protein G Dynabeads (Novex Lifetech) was washed 3 times in block solution and then resuspended in 500 µl of block solution. Beads were then incubated with 5 µl of antibody (H3K27me3(Millipore 07449), IgG (Santa Cruz Biotech sc-2027)) and incubated at 4 oC for 4 hours on a rotator. Beads were then washed 3 times in 500 µl block solution and supernatant removed. For ChIP libraries, the NEBNext ChIPSeq library kit or the SimpleChIP® Plus Enzymatic Chromatin IP Kit was used as per instructions. For WGBS libraries, DNA was bisulfite treated using the Zymo EZ DNA Methylation Lightning Kit followed by library preparation using the TruSeq DNA Methylation Kit (Illumina).