title: GSM6009022: CTCF ChIP-seq on embryonic 13.5 day mouse forebrain, biological replicate 2; Mus musculus; ChIP-Seq
library_construction_protocol: ~30 mg fixed tissues were thawed on ice and the nuclei preparation was performed using truChIP® Chromatin Shearing Kit (Covaris) following the manufacturer's instruction. The lysed nuclei were sheared using Covaris M220 with following setting: Setting: power, 75 W; duty factor, 8%; cycle per burst, 200; time, 12 min; temp, 7 °C. The sheared chromatins were cleared by centrifugation at 15,000×g for 12 min. The supernatant was collected and incubated with M-280 sheep anti-rabbit magnetic beads (Thermo Fisher, 11203D) for 3 hours at 4°C with slow rotation for preclearing. ~5% of precleared cell lysate was saved as input control after the incubation was done. The rest of precleared lysate was mixed M-280 sheep anti-rabbit magnetic beads that coupled with 0.9 μg of CTCF antibody (3418, Cell Signaling) and rotate at 4°C for 16 hours. On the next day, the beads were collected using magnetic stand and the supernatant was discarded. The beads are then washed with RIPA buffer three times, high-salt RIPA buffer (10 mM Tris, pH 8.0, 300 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.1% sodium deoxycholate) twice, LiCl buffer (10 mM Tris, pH 8.0, 250 mM LiCl, 1 mM EDTA, 0.5% IGEPAL CA-630, 0.1% sodium deoxycholate) once, TE buffer (10 mM Tris, pH 8.0, 0.1 mM EDTA) twice. Washed beads were treated with 10 μg Rnase A in extraction buffer (10 mM Tris, pH 8.0, 350 mM NaCl, 0.1 mM EDTA, 1% SDS) for 1 hours at 37°C, followed by reverse crosslinking in the presence of proteinase K (20 μg) overnight at 65°C. After reverse crosslinking the DNA was purified and quantified. The input control DNA was reverse crosslinked and purified in the same way as ChIPed DNA as described above. Library preparation was performed using QIAseq Ultralow Input Library Kit (Qiagen) starting from 10–100 ng ChIPed DNA or input DNA. After PCR the amplified libraries were purified with SPRI Beads to extract fragments between 200-600bp for sequencing.