GSM6631098: H3K4me1_hPGC_wk8_F_r1; Homo sapiens; ChIP-Seq
Cells were thawed on ice, incubated with 2 µl of 1 % Triton X-100, 1 % Sodium deoxycholate and digested with Micrococcal Nuclease (MNase) (NEB). MNase activity was blocked by addition of 11 µl 100 mM EDTA and cell lysate was incubated for 1 h in 400 µl of IP buffer at 4 °C followed by 2 h incubation in the presence of 5 µl blocked protein A/G beads (blocking buffer: 100 µg/ml yeast tRNA, 0.1 % BSA in IP buffer). After the removal of the protein A/G beads, the pre-cleared cell lysate was added to the antibody bead complex (antibody was incubated with 5 µl blocked protein A/G beads for 3 h on 4 °C) overnight at 4 °C. Unbound chromatin was removed, and beads were sequentially washed for 4 min for 1.) two times with low salt wash buffer, 2.) two times with high salt buffer, and 3.) two times with LiCl wash buffer (20 mM Tris-HCl pH 8.0, 2 mM EDTA, 250 mM LiCl, 1 % NP-40, 1 % Sodium deoxycholate). To elute the bound DNA, beads were incubated in Proteinase K digestion buffer (20 mM HEPES pH 8.0, 1 mM EDTA, 0.5 % SDS, 1 mg/ml RNase, 0.4 mg/ml Proteinase K) for 15 min at 55 °C and 1 h at 65 °C. The DNA was purified from the eluate through AMPure XP beads following the manufacturer's instructions and eluted in 20 µl EB buffer (MinElute Reaction Cleanup Kit; Qiagen). ULI-NChIP-seq libraries were generated by the KAPA Hyper Prep Kit (KAPA Biosystems) according to manufacturer's protocol. To minimize adaptor dimer formation, the NEBNext Adaptor and NEBNext Index PCR Primers from the NEBNext® Multiplex Oligos for Illumina (Index Primers Set 1) (NEB, E7335S) were used. After library amplification, libraries were purified by AMPure XP beads with double-sided size selection. The libraries were subjected to paired-end sequencing on HiSeq 4000 sequencing system (Illumina)