GSM5322397: K562_ChIPseq_ORC2_HU047_LY111d; Homo sapiens; ChIP-Seq
For ORC2 ChIP-seq, the supernatants were pre-cleared by 40 µL protein G dynabeads for 1 hour and then incubated with 40 µL Streptavidin T1 beads (Dynabeads) for 6 hours at 4℃. Beads were harshly washed by freshly-made 2% SDS, high-salt buffer (500 mM NaCl; 0.1% SDS; 1% Triton X-100; 2 mM EDTA; 20 mM Tris-HCl, pH 8.0), LiCl buffer (0.25 M LiCl; 1% NP-40; 10 mM Tris-HCl; pH 8.0, 1 mM EDTA) and TE buffer. The beads-bound chromatin was eluted with the elution buffer (20 mM Tris-HCl, pH 8.0; 10 mM EDTA; 1% SDS) at 65℃ overnight. For MCM5 ChIP-seq, the soluble fractions were directly applied to anti-MCM5 (1:50) enrichment at 4℃ overnight. Then protein G dynabeads were added and incubated for 1 hour. Beads were washed as those of ORC2/5 ChIP, except that the wash with 2% SDS was removed. Chromatin on beads was eluted twice with elution buffer at 65℃ for 15 min.   The eluate fraction was digested by RNase A and incubated with Proteinase K overnight. DNA was purified, end-repaired, and poly-dC tailing as described [72]. After poly-dC tailing, DNA was tagged with a biotinylated primer, captured by Streptavidin C1 beads, and ligated with an indicated bridge adapter (see the resource table) overnight at room temperature. The ligated products were then washed and tagged with Illumina P5-I5 and P7-I7 sequences via PCR for 13 cycles. Sequencing was performed on the Illumina Hiseq platform (2 × 150 bp).