GSM9008916: K562, CTCFi, H3K9ac, ChIP, rep1; Homo sapiens; ChIP-Seq
For ChIP assay, nuclei were digested with 100 units of MNase at 37°C for 15 min. Mono or di-nucleosomes sized chromatin was incubated with antibodies for 3 h at 4°C and recovered with protein A agarose beads. Spike-in chromatin (Active Motif; 25 ng for CTCF and 50 ng for the others) and spike-in antibody (Active Motif; 1 μg for CTCF and 2 μg for the others) were included as internal controls. RNA was extracted using Rneasy Mini Kit (Qiagen). Ribosomal RNAs (rRNAs) were removed using Invitrogen RiboMinus Transcriptome Isolation Kit (human/mouse) (Invitrogen). 125 ng of rRNA-removed RNA was used for the construction of sequencing libraries. ChIP DNA (0.5 ng for CTCF, 1 ng for H3K9ac, and 10 ng for H3K9me2 and H3K9me3) and input DNA (10 ng) were processed using NEBNext Ultra II DNA Library Prep Kit (New England Biolabs) with manufacturer's instructions. The ends of ChIP DNA were repaired by NEBNext Ultra II End Prep enzyme mix and ligated with NEBNext adaptors. Adaptor-ligated DNA was selected at 175 bp size using NEBNext sample purification bead and amplified using the adaptor primers. The fragments are purified using NEBNext sample purification beads. RNA-seq libraries were prepared using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB) following manufacturer's protocols.