GSM3261527: HeLa_GWPBS-Cap-400 mM; Homo sapiens; OTHER
For GWPBS-Cap samples, lysates were washed by cold NaCl Buffer containing different concentrations (50 mM,100 mM, 200 mM, 400 mM, 1000 mM or 2000 mM) of NaCl. After 2 hours' rotation, nuclei were pelleted and resuspended in the same cold NaCl Buffer. Formaldehyde solution were added to cross-link protein and DNA and the complexes were sheared by sonication. Supernatants containing the solubilized chromatin DNA: protein complexes were clarified and added TCEP to reduce disulfide bonds of proteins. These complexes were isolated with active Amine-coated beads through sulfo-SMCC. For MNase-seq samples, lysates were clarified from MNase treated nuclei. For RNA-seq samples, total RNA was extracted using TRIzol and an aliquot of the RNA was reversed transcribed. For GWPBS-Cap samples, DNA was end-repaired using T4 Polynucleotide Kinase. Phosphorylated 5' ends were treated with Lambda exonuclease and 3' single-stranded Sp6 adapters were ligated using T4 RNA Ligase 1. The DNA was extracted and double-stranded DNA was built using Klenow Fragment. DNA was added A-tail using dATP and Klenow Fragment (3'→5' exo-) and T7 double-stranded adapters were ligated to ends of DNA using T4 DNA Ligase. After adapter ligation DNA was PCR amplified with Sp6 and T7 primers for 20 cycles and library fragments of 80~120 bp (insert plus PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured and the paired-end sequencing was performed on Illumina Hiseq 4000 platform. For MNase-seq samples, The extracted 147 bp DNA bands were separated on agarose gel. The library  preparation and the high-throughput sequencing was performed on Illumina HiSeq 4000 platform. For RNA-seq samples, RNA libraries were prepared for sequencing using standard Illumina protocols.
GEO Accession
GSM3261527