title: GSM6925410: LSD1_HCT116_WT_H3K4me3_rep2; Homo sapiens; ChIP-Seq
library_construction_protocol: 5x 106 cells were cross-linked with 1% formaldehyde for 10 minutes and quenched by 125mM glycine for 5 minutes at room temperature with gentle shaking, and washed twice with PBS. Cells were then pelleted and lysed in ice-cold lysis buffer1 (50mM HEPES-KOH, pH 7.5, 140mM NaCl, 1mM EDTA, 10% Glycerol, 0.5% NP-40, 0.25% Triton X-100) and lysis buffer 2 (10mM Tris-HCl pH 8.0, 200mM NaCl, 1mM EDTA, 0.5mM EGTA) supplemented with cOmplete protease inhibitors (Roche) for 10 minutes. The cell lysate was sonicated using Covaris M220 with 5% duty factor for 10min at 4 °C to shear DNA to fragments (200~1000 bp). Soluble chromatin was diluted in shearing buffer (1mM EDTA, 10mM Tris-HCl pH 8.0, 0.1% SDS) with 1% Triton X-100 and 150 mM NaCl and incubated with 3μg ChIP-grade antibody at 4°C overnight with gentle shaking. 5% of input was stored prior to the de-crosslinking procedure. 30μl Protein-G magnetic beads (Thermo Fisher Scientific, 01134323) was used for subsequent pull-down of antibody-chromatin complex by incubatioin for 2 hours at 4°C with gentle shaking. The beads were washed with following buffers for 2 times each: IP buffer (0.1% SDS, 1% Triton X-100, 1mM EDTA, 150mM NaCl, 10mM Tris-HCl pH 8.0), High Salt Wash Buffer (0.1% SDS, 1% Triton X-100, 2mM EDTA, 500mM NaCl, 20mM Tris-HCl pH 8.0), LiCl Wash Buffer (250mM LiCl, 1% NP-40, 2mM EDTA, 10mM Tris-HCl pH 8.0), and TE buffer+50mM NaCl (1mM EDTA, 50mM NaCl, 10mM Tris-HCl pH 8.0) 1 times. DNA was eluted with Elution Buffer (1% SDS, 10mM EDTA, 50mM Tris-HCl pH 8.0) at 65℃ for 30 minutes with shaking at 1,400 rpm. The supernatant was then collected and incubated at 65°C for overnight to reverse-crosslink the DNA. The day after, the enriched DNA was treated with 4 µL of 20 mg/mL RNase at 37 °C for 2 h and 4 µL of 20 mg/mL Proteinase K (MIKX, FZ690) at 55°C for 2h. The immunoprecipitation DNA was purified by phenol-chloroform-isoamyl alcohol (Solarbio, P1012-100), washed with ethanol, eluted in nuclease-free water, and used for subsequent experiments. The following antibodies were used in ChIP: H3K4me1 (Abcam, ab8895), H3K4me3 (Abcam, ab8580) Sequencing libraries were constructed and sequenced by the Novogene Bioinformatics Institute (Novogene, Beijing, China).