GSM2704160: minus DOX; Homo sapiens; RNA-Seq
For chromatin immunoprecipitation, differentiating day 5 cells were harvested using Accutase, resuspended in ITS/HSA-containing medium and fixed for 10 min by adding freshly thawed formaldehyde solution to a final concentration of 1% (w/v). After stopping with 125 mM glycine, pellets of 5*106 cells were stored at -80°C or processed immediately. Samples were lysed in hypotonic Tris buffer and sonicated 35 times with a 30 s on / 30 s off cycling protocol using a Diagenode Bioruptor device at high power (volume: 300 µl), to an average DNA fragment size of ~200 bp. Following 10-fold dilution with 50 mM Tris pH8 / 200 mM NaCl / 5 mM EDTA / 0.5 (v/v) NP40, sheared samples were incubated with 10 µg of antibody over night at 4°C on a rotator. The antibody used was raised against a transgenic ISL1-HA tag. Antibody/chromatin complexes were isolated using Pierce protein A/G-conjugated magnetic beads (Thermo # 26162) and washed repeatedly with high-salt (500 mM NaCl) wash buffer. ChIP samples were eluted using 65°C 1% (w/v) SDS-containing buffer, crosslink-reversed in the presence of 200 mM NaCl for 4 h at 65°C, treated with RNase A and Proteinase K, and purified on Qiagen PCR purification columns. Illumina kit # IP-202-1012 following the manufacturer's instructions Pooled starting material from independent experiments