GSM5839644: PTC11_2_H3K27me3; Mus musculus; ChIP-Seq
Chromatin immunoprecipitation was performed on two PTC iPSC lines and two TTF iPSC lines following the protocol outlined in the SimpleChIP Enzymatic Chromatin IP Kit (Cell Signaling Technologies, 9003). Briefly, 30-60 million cells were fixed in 1% methanol-free formaldehyde (Thermo Fisher, 28906) at room temperature (RT) for 10 min. Glycine was added to the fixed cells at a final concentration of 0.125 M for 5 min at RT. Cells were centrifuged at 500 g, 4℃, 5 min, and washed twice with ice cold phosphate buffered saline (PBS). Chromatin was digested with micrococcal nuclease at a concentration of 0.1 µl/million cells for 30 min at 37℃. Cells were then sonicated using the Sonics VibraCell (VCX130) with 3 mm probe at 40% amplitude and 5 sets of 20 s pulses. Approximately 10 µg of digested, cross-linked chromatin was incubated with 10 µg of anti-H3K4me3 (Abcam, AB8580, lot #GR3175719-3), anti-H3K27me3 (Millipore, 07-449, lot #3018864), or anti-H3K36me3 (Abcam, AB9050, lot #GR3210077-1) antibody overnight at 4℃. ChIP-grade protein G magnetic beads were added to each immunoprecipitation reaction and incubated for 2 h at 4℃. Magnetic beads were pelleted using a magnetic separation rack (Cell Signaling Technologies, 7017) and washed in a sequence of low salt and high salt washes. Finally, chromatin was eluted from the magnetic beads and DNA was reverse cross-linked from the histone. DNA was cleaned up using the Qiaquick PCR Purification Kit (Qiagen). Enrichment was validated using qPCR with Pax2 primers (Active Motif, #71020) and Actb2 primers (Active Motif, #71017). Libraries were prepared according to the manufacturer's instructions, by the Centre for Applied Genomics (Toronto, ON) on the Illumina HiSeq 2500 and 50 million reads were acquired for each sample.