GSM6600915: H3K9me3_EmbryoidBody_WT_rep1; Mus musculus; ChIP-Seq
Trypsinized mESCs were fixed and crosslinked using 1% paraformaldehyde (diluted in PBS) for 10 minutes. The crosslinking was then quenched using 1 mM of Glycine for 10 minutes. The mESCs were then lysed and sonicated using the M220 ultrasonicator (Covaris) in 1 ML tubes with an AFA Fiber and the following conditions ( cycles/burst = 200, Duty Factor = 20%, Peak Intensity Power = 75, time = 10 minutes and temperature = 7 degrees celcius). The sheared DNA was then pre-cleared using magnetic beads for 1 hour. Antibodies were then added to the DNA and incubated overnight at 4 degrees. Magnetic beads were added to each samples. The DNA samples were then eluted and de-crosslinked overnight at 62 degrees. Residual RNA and protein was then removed using RNAseA and proteinaseK and DNA was isolated using the Qiagen PCR purification Kit using manufacturer's instructions. For ChIP samples that used CUT&RUN (L3MBTL2) was used using manufacturer's instructions (EpiCypher CUTANA ChIC/CUT&RUN Kit, 14-1048) The eluted DNA from the CUT&RUN or ChIP was used to construct multiplex libraries with the NEBNext Ultra II DNA library Kit using manufacturer's instructions.