GSM8705149: N2A, ADA-Clone1, RAD21, rep2, ChIP-seq; Mus musculus; ChIP-Seq
Briefly, cortical tissues were dissected out using fine forceps and immediately transferred to a dish containing ice-cold NeuroBasal medium. The tissues were then torn into pieces using fine forceps and transferred to an Eppendorf tube containing 0.25% trypsin-EDTA. The sample was incubated at 37°C for 15 min with continuous shaking at 300 rpm. The trypsin digestion of tissues was quenched by the addition of 10% FBS in the PBS buffer. After filtering the dissociated tissues into a single-cell suspension, the volume was adjusted to 5 ml using 10% FBS in the PBS for crosslinking. The purified DNA was subjected to high-throughput library preparation following the protocol recommended by the manufacturer. Briefly, 10 ng of purified DNA was used as a template and combined with End Prep Mix on ice. The reaction was incubated using the following program (20°C, 15 min; 65°C, 15 min). Next, the sample was directly proceeded to adapter ligation and mixed with Rapid Ligation buffer, Rapid DNA ligase, and Adapter-HTS. The sample was then incubated at 20°C for 15 min. Next, the ligated DNA was purified using well-mixed AMPure XP Beads. The DNA purification process was consistent with that described in RNA-seq. Finally, the purified product was amplified using the following mixture (P5 primer, P7 primer, and Amplification Mix ) and proceeded to PCR (95°C, 3 min; 98°C, 20 s, 60°C, 15 s, 72°C, 30 s for 11 cycles; and a final extension at 72°C, 5 min) with P5 and P7 primers for sequencing. The PCR products were also purified using well-mixed AMPure XP Beads. All ChIP-seq libraries were sequenced on an Illumina NovaSeq 6000 platform.