GSM7042606: ChIP_WT_CTCF_rep9; Mus musculus; ChIP-Seq
2 × 106 of cortex neurons or cultured cell were used for one experiment. Neuron cells were first dissected and digested using 0.00625% collagenase. The neuron suspension or cultured cell were cross-linked using 1% formaldehyde in 10% FBS/PBS for 10 min at room temperature followed by washing twice with PBS solution containing proteinase inhibitors (Roche). Cells were then resuspended and lysed twice with 1ml prechilled lysis buffer containing proteinase inhibitors (Roche) for 10 min with slow rotations. For neuron sample, additional SDS was then added to the lysis solution to adjust final SDS concentration to 0.4% to help sonication. Cells were then sonicated to break DNA fragments to about 100-10000 bp using the Bioruptor system (25% energy, with working time 15s and resting time of 45s, 20cycles). After sonication, lysis buffer not containing SDS was added to adjust final SDS concentration to 0.1% to allow binding of antibodies to proteins while cultured cell move directly to the antibody binding step. After removing the insoluble debris by centrifuging at 14000g for 10 min, the lysate was precleaned with protein-A agarose beads (16-157; Millipore) for 1hr with slow rotation to remove non-specificity binding and incubated with specific antibodies overnight with slow rotation at 4°C. The next day the antibody-protein-DNA complex was purified by adding protein A-agarose beads and incubate for 4hr with slow rotation, followed by washing with low salt buffer, high salt buffer, no salt buffer, LiCl buffer and TE buffer and finally eluted into elution buffer (0.1M NaHCO3, 1% SDS). Reverse cross-link and DNA purification was performed and the sample were prepared into libraries using the VAHTS Universal DNA Library Prep Kit for Illumina V3 (Vazyme) following the manufacturer's instruction. The ChIP-seq libraries were sequenced using an Illumina Novaseq6000 platform.