GSM7029334: ChIP_H1_YTHDC2WT_H3K27me3_1; Homo sapiens; ChIP-Seq
Cells were crosslinked with the crosslinking buffer (10mM NaCl, 0.1 mM EDTA, 0.05 mM EGTA, 5 mM Hepes pH8.0, 1% formaldehyde) at room temperature for 10 min. The crosslinking reaction was stopped by adding 2.5M glycine to a final concentration of 0.125M and incubation at room temperature for another 5 min. Cells were washed with ice cold PBS twice and transferred to 15 mL tubes in PBS. After centrifugation at 500g for 5 min, the PBS was discarded. Cell pellets were resuspended in 50 μl lysis buffer (1% SDS, 50 mM Tris-HCl, pH 8.0, 20 mM EDTA supplemented with 10x protease inhibitor) and incubated for10 min on ice. The cell lysates were diluted to 500 μl with the cold TE buffer. The lysates were sonicated using Covaris M220. The sonicated lysates were centrifuged at 12000 g for 15 min at 4 °C. The supernatants were transferred into new tubes and DNA concentrations were measured on a NanoDrop. For each replicate, 20 μg of chromatin was used. To pre-clear the chromatin solutions, 20 μl lgG Dynabeads (Life Technologies, 11204D) were washed and added. The chromatin beads solutions were rotated gently in the cold room for 2 hours. After the incubation, place tubes on a magnetic stand to collect beads. 100 μl supernatants were transferred into new tubes. 100 μl Binding Buffer (1% Triton-X, 01% Sodium Deoxycholate supplemented with protease inhibitor in TE buffer) was added to the 100 μl chromatin. The mixture was incubated in the cold room overnight on a rotator. Next day, 20 μl lgG pre-washed Dynabeads were added into each reaction and incubated for another 2 hours. After incubation, the beads were washed five times with wash buffer (50 mM Hepes, pH 8.0, 1% NP-40, 0.70% Sodium Deoxycholate, 0.5 M LiCl, 1 mM EDTA supplemented with protease inhibitor) and twice with cold 1xTE. After the final wash, 150 μl ChIP elution buffer (10 mM, Tris-HCl pH 8.0, 1 mM EDTA, 1% SDS) were added into tubes. The beads mixture was transferred to new tubes and incubated at 65°C for 20 minutes at 1300 rpm on a Thermomixer. The supernatants were transferred into tubes and incubated at 65°C overnight to reverse crosslinks. Next day, 250μL1xTE and 8 μl of 10 mg/mL RNase A was added to each sample and incubated at 37°C for 1 hour. 8 μl of 20 mg/mL Proteinase K was added and incubated at 55°C for 1 hour. After that, the DNA was purified and measured on a NanoDrop. To generate ChIP libraries, DNA ends were repaired. ployA tails were added to the ends and then adaptors were ligated at room temperature. Last, the DNA was amplified with TruSeq primers and purified with magnetic beads. Libraries were sequenced on the MGISEQ-2000 platform in pair-end mode with 100 base pair (bp) per read.