GSM4847908: Bru-seq Rep 2 G2; Homo sapiens; OTHER
For RNA-seq and Bru-seq, live cells were stained with Hoechst 33342 (Cat #B2261, Sigma-Aldrich), and then sorted by fluorescence-activated cell sorting (FACS) to obtain cell fractions at the corresponding cell cycle phases G1, S, and G2/M. For cells used in construction of Hi-C libraries, cells were crosslinked with 1% formaldehyde, the reaction was neutralized with 0.125 M glycine, then cells were stained with Hoechst and sorted into G1, S, and G2/M fractions. Cross-linked chromatin was digested with the restriction enzyme MboI for 12 hours. The restriction enzyme fragment ends were tagged with biotin-dATP and ligated in situ. After ligation, the chromatins were de-cross-linked, and DNA was isolated for fragmentation. DNA fragments tagged by biotin-dATP, in the size range of 300-500 bp, were pulled down for sequencing adaptor ligation and polymerase chain reaction (PCR). RNA-seq library construction was carried out in the University of Michigan DNA Sequencing Core facilities facility, and sequence reads of 50-base in length were generated on an Illumina HiSeq 2500 station. We converted the Bru-labeled transcripts from the different samples into cDNA libraries and they were deep-sequenced at 50-base length on an Illumina HiSeq2500 station. The Hi-C PCR products were sequenced on an Illumina HiSeq2500 station generating reads of 125-base in length, paired-end.