GSM7035840: E14 ESCs 2,5HD-treated, RING1B, Cell Signaling, biological replicate 2; Homo sapiens; Mus musculus; ChIP-Seq
Pellets were then thawed and combined with 1×10^6 formaldehyde-fixed human MCF-7 cells (for downstream calibration of ChIP-seq data). Following lysis, cells were sonicated using a cooled Bioruptor (50 cycles, 1 minute cycles of 30s on/30s off on “high” setting at 4°C). The sonicated extract was pre- cleared by centrifugation at 16,000g for 10 min at 4°C. The supernatant was transferred to a fresh tube and supplemented with BSA to a final concentration of 25 mg/mL. A sample of the chromatin was retained as an input reference.   Antibodies were precoupled to a 1/1 mixture of protein A (Life Technologies 10001D) and protein G (Life Technologies 1003D) Dynabeads at a ratio of 1 mg antibody per 30 mL of Dynabead suspension by rotation for 2 hours at 4°C. Cell equivalents (~6.5×10^6) of lysate were added to 7.5 microgrammes of anti-Ring1B (Cell Signaling D22F2), 5 microgrammes of anti-H3K27me3 (Cell Signaling C36B11), or 5 microgrammes Rabbit IgG (Cell Signaling) respectively, and incubated overnight on a rotating wheel at 4°C. Following incubation, bead-associated immune complexes were washed sequentially with ChIP dilution buffer, wash buffer A, and wash buffer B, each for 10 min at 4°C on a rotating wheel, followed by two washes in TE buffer at room temperature. Chromatin was released by incubating the beads in 100 µL of elution buffer (0.1M NaHCO3, 1% SDS) for 15 min at 37°C, followed by the addition of 20 µg of RNase A and 6 µL of 2 M Tris (pH 6.8) and incubation for 1 h at 37°C and finally by the addition of 20 µg proteinase K and incubation overnight at 65°C to degrade proteins and reverse the cross-links. Dynabeads were removed using a magnetic rack and the chromatin purified using PCR purification columns (Qiagen) according to the manufacturer's instructions. Libraries (Ring1B and corresponding input samples only) were constructed using the NEBNext Ultra II DNA library preparation kit for Illumina according to the manufacturer's instructions (NEB E7645S). To determine the number of PCR cycles required for amplification, one aliquot of library preparation from each sample were supplemented with evagreen so that amplification could be monitored by quantitative PCR on a BioRad C1000 Touch Thermal Cycler. To allow for sample multiplexing, PCRs were performed using index primers (NEBNext multiplex oligos for Illumina, set 1, E7335 and amplified to linear phase). Size selection purifications following the ligation and amplification PCR steps were performed with 0.9× reaction volumes of Agencourt AMPure XP beads (Beckman Coulter A63880). Purified libraries were combined as a 8-sample equimolar pool containing a combination of indexes from 1–12 and sequenced on an Illumina NextSeq on an Illumina NextSeq 2000 on a P2 flow cell (paired-end 75-bp reads).