GSM6030893: PFA2-Rx-XChIP_K27me2; Homo sapiens; ChIP-Seq
Reverse cross linking took place on a heat block at 65°C for 4 hr. ChIP samples were then treated with 2ul RNase Cocktail at 65°C for 30 min followed by 2ul Proteinase K at 65°C for 30 min. Samples were then purified with QIAGEN MiniElute PCR purification kit as per manufacturers' protocol. In parallel, input samples (chromatin from about 50,000 cells) were reverse crosslinked and DNA was isolated following the same protocol. Library preparation was carried out using Kapa HTP or HyperPrep Illumina library preparation reagents. Ffor HTP kit, 25ul of ChIP sample was incubated with 45ul end repair mix at 20°C for 30 min followed by Ampure XP bead purification. A tailing: bead bound sample was incubated with 50ul buffer enzyme mix for 30°C 30 min, followed by PEG/NaCl purification. Adapter ligation: bead bound sample was incubated with 45ul buffer enzyme mix and 5ul of different TruSeq DNA adapters (Illumina) for each sample, at 20°C for 15 min, followed by PEG/NaCl purification (twice). Library enrichment: 12 cycles of PCR amplification. For HyperPrep kit, end-repair and A tailing were performed in one reaction: 15ul of ChIP sample was incubated with 45ul end repair+A-tailing mix at 20°C for 30 min, then 65°C for 30 min. Directly proceeded to adapter ligation by adding 5ul of IDT for Illumina Unique Dual Indexes (IDT) adapters and 45ul ligation buffer enzyme mix, incubating at 20°C for 15 min, followed by Ampure XP bead purification. Library enrichment: 10 cycles of PCR amplification. Size selection was performed after PCR using a 0.6x/0.8x ratio of Ampure XP beads (double size selection) set to collect 250-450bp fragments.