GSM7324746: IP_1; Homo sapiens; ChIP-Seq
Approximately 1 × 10^7 cells were crosslinked in 1% Formaldehyde for 10 min and stop the reaction by 0.125 M glycine for 5 min. The crosslinked cells were lyses in Lysis buffer (1× PBS, 0.1% SDS, 0.5% NP-40 and 0.5% sodium deoxycholate) and sonicated to generate DNA fragments for 200-1000 bp. For immunoprecipitation, Protein-DNA complex was immunoprecipitated by incubating with ChIP-grade Protein A/G Magnetic Beads (26162, Invitrogen, USA) conjugated with anti-FLAG antibody (F7425, sigma, USA), or IgG (B900610, Proteintech, China) at 4℃ for 2h. The beads were washed with LiCl Immune Complex wash buffer (0.5 M LiCl) for five times, TE buffer (10Mm Ttis (PH 8.0), 1mM EDTA (PH 8.0)) for one time, respectively. Resuspend the beads with 100ul Elution Buffer (100 mM NaHCO3 ,1% SDS) and reverse cross-linked by overnight incubation at 65 ℃. After sequential RNase A and proteinase K treatment, DNA fragments were purified by phenol extraction and ethanol precipitation. The libraries were performed by using VAHTS Universal DNA Library Prep Kit for Illumina V3 (ND607, Vazyme, China) according to manufacturer's instructions and PCR products corresponding to 200-500 bps were enriched, quantified and finally sequenced on Novaseq 6000 sequencer (Illumina, USA) with PE150 model.