GSM4766799: HeLa_Input_No treatment; Homo sapiens; OTHER
Cells were washed with ice-cold PBS and then fixed for 12 minutes at RT. Fixed cells were permeabilized, clicked, resuspended in lysis buffer and sonicated with Bioruptor with the following setting: 30 seconds on, 30 seconds off, 30 cycles. After reserving an aliquot for input, dilution buffer (1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris-HCl [pH 8.1], 3 mM CaCl2) at 10 times the volume of lysis buffer was added and mixed well, and biotinylated DNA was pulled down using M-280 streptavidin Dynabeads. After being extensively washed, the beads were resuspended in 0.1 × TE (10 mM Tris-HCl pH [8.0], 1 mM ethylenediaminetetraacetic acid), and RNase A was added and incubated at 37℃ for 60 min. SDS was added to a final concentration of 1% and proteinase K (Thermo Fisher Scientific) was added to a final concentration of 20 mg per ml, and the suspension was incubated at 55℃ for 2 h and then 65 ℃ overnight to reverse cross-links and degrade proteins. After incubation, phenol-chloroform-isoamyl alcohol extraction was performed, and the DNA in the aqueous phase was precipitated using 70% isopropanol, 30 mM sodium acetate, and 150 mg/ml glycogen at -80℃ for at least 30 min followed by centrifugation at 4℃, 16,000 × g for 30 min. The DNA pellet was washed with 75% ethanol and resuspended in 20 ml TE buffer. Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit (Part# 0801-0303). Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3' end. After adapter ligation DNA was PCR amplified with Illumina primers for 15 cycles and library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Illumina HiSeq 2500 following the manufacturer's protocols.