GSM8750984: H3K4me3_DMSO4h; Mus musculus; ChIP-Seq
To harvest, medium was removed and plates were washed 2x with PBS before incubation for 10 minutes in 1% formaldehyde. Plates were then washed 2x with PBS and cells scraped and transferred to a 50ml tube in PBS. Cell pellets were centrifuged for 5min at 1000xg, PBS removed, and the cell pellets flash frozen in liquid nitrogen. Pellets were stored at -80C until further use. Nuclei extraction was performed according to PMID: 34363071, and sonication was performed on a Covaris E220 Evolution with 10% duty factor. Chromatin sonication was validated using a TapeStation and total chromatin was quantified by Qubit. ChIP-seq spike-in was prepared from Drosophila S2 chromatin and added to 1% of the final chromatin input for each ChIP. RNA Pol II, RAD21, RING1B, and SUZ12 ChIPs were performed with 50ug, H2BUb and H2A.Z with 25ug, and H3K4me3, H3K36me3, H3K27ac, H3.3, H3, and H2B ChIPs were performed with 10ug of input chromatin. Chromatin was incubated with antibodies overnight at 4C before incubation with Protein A/G beads for 3h at 4C. For histone ChIPs, beads were rinsed 2x with low-salt (150mM) RIPA buffer, washed 1x with low-salt RIPA buffer for 5min, washed 3x with high-salt (500mM) RIPA, and 1x in LiCl wash buffer before being eluted in 1x TE. For non-histone ChIPs, 2x low-salt rinses, 2x low-salt washes, 1x high-salt wash, and 1x LiCl wash were performed. Libraries were costructed using the KAPA Hyperprep kit and Illumina-compatible indexed adapters (IDT, 10005903) following manufacturer's instruction. For the amplification step, 6-7 PCR cycles were used. Subsequently, the DNA underwent size selection using Agencourt AMPure XP beads (Beckman Coulter, A63881) to obtain fragments between 200-700 bp. Libraries were validated using D5000 DNA ScreenTape on a TapeStation 4200.