GSM5963434: CD69negDP_H3K27ac_ChIPseq_rep1; Mus musculus; ChIP-Seq
Cells were cross-linked in 1% formaldehyde for 10 min at room temperature and then the reaction was quenched with 125 mM glycine at room temperature for 5 minutes. [Hi-C] Chromatin was digested with 100 units of MboI overnight at 37 °C while shaking at 900 rpm. After overnight incubation, 100 units of MboI were added for the second-round digestion for 2-4 hours. The DNA polymerase I, large (Klenow) fragment, was used to fill in MboI-digested fragment overhangs in the presence of biotin-labelled dATP. Fragment ends were then ligated with 4000 units of T4 DNA ligase by incubating at room temperature for 4-6 h with rotation at 12 rpm. After ligation, cell nuclei were collected by centrifugation, and chromatin was reverse crosslinked overnight at 65 °C. RNase A was used to remove RNA after decrosslinking. Genomic DNA was isolated and precipitated by sodium acetate-ethanol precipitation, and then purified DNA was sheared for 8 min using a sonicator (Bioruptor) with high power setting, and 30 seconds on and 30 seconds off per minute. DNA fragments in the range of 300–500 bp were then selected using the AMPure XP beads (Beckmann Coulter). [ChIP-seq] Cell nuclei were isolated using a RIPA lysis buffer (10mM Tris pH7.5, 1mM EDTA, 1% Triton X-100, 0.1% Sodium deoxycholate, 0.1% SDS, 0.15M NaCl, 1X Roche protease inhibitors). Following isolation of cell nuclei, genomic DNA was sonicated in a high-salt RIPA buffer (10mM Tris pH7.5, 1mM EDTA, 1% Triton X-100, 0.1% Sodium deoxycholate, 1% SDS, 0.8M NaCl, 1X Roche protease inhibitors) for 30 minutes on a sonicator (Bioruptor) with high power setting, and 30 seconds on and 30 seconds off per minute. Immunoprecipitation was performed in a RIPA buffer (10mM Tris pH7.5, 1mM EDTA, 1% Triton X-100, 0.1% Sodium deoxycholate, 0.1% SDS, 0.15M NaCl, 1X Roche protease inhibitors), and then antibody-antigen complex was precipitated using Dynabead Protein G (Thermo Fisher). Protein was digested using Proteinase K, and then DNA-protein complex was reverse crosslinked overnight at 65 °C. RNase A was added to remove RNA after decrosslinking. [Hi-C] After biotin-labelled DNA fragments were captured on Dynabeads MyOne Streptavidin T1 (Thermo Fisher), fragment ends were repaired in a mixture of enzymes containing T4 polynucleotide kinase, T4 DNA polymerase I and DNA polymerase I, large (Klenow) fragment, and then NEBNext adaptors for Illumina sequencing were ligated to the dA-tailed fragment ends. After the USER enzyme digestion, NEBNext oligos for Illumina sequencing were used for PCR for library preparation. A PCR titration was firstly carried out to determine the lowest number of PCR cycles (8-10 cycles in this work). Final Hi-C sequencing libraries were quantified and checked for size distribution using Bioanalyzer (Agilent Technologies), and then sequenced on an HiSeq 2500 sequencer (Illumina). [ChIP-seq] ChIP-seq library preparation were carried out using the NEBNext ChIP-Seq Library Prep Master Mix Set for Illumina (NEB) according to the manufacturer's protocol.