GSM6585948: H3K27ac Chip-Seq of Mus musculus: ESCs-WT-input; Mus musculus; ChIP-Seq
For RNA-seq: cells were lysed with Trizol reagent (Life Technologies) and RNA was extracted according to the manufacturer's instructions. For Chip-seq, 10 M cells cells were crosslinked with 1% formaldehyde (methanol free) in DMEM at a volume of 1 mL of formaldehyde for every one million cells. Cells were cross-linked 10 min at room temperature with rotation. Glycine was added into a final concentration of 125 mM to quench the formaldehyde. Samples were incubated 15 min on ice and then spun down at 2500 g for 5 min. The supernatant was discarded, and pelleted nuclei were washed once with 500 μL pre-chilled cell Lysis Buffer (10mM Tris-HCl pH 8.0, 10mM NaCl, 0.2% NP-40, 1×protease inhibitors) and then rotated at 4°C for 30 min. Samples were then spun down at 2500 g for 5 min and the supernatant discarded, while pelleted nuclei were washed twice with 500 μL of ice-cold ChIP Lysis Buffer. The volume of pelleted material was brought up to 1mL in Nuclear Lysis Buffer (50mM Tris-HCl pH 7.5, 10 mM EDTA, 1% SDS, 1×protease inhibitors) and incubated 5 min at room temperature. Samples were then divided equally into 3 parts, sheared for 30 cycles (30 s “ON” and 30 s of “OFF”), and then spun down at 16100 g for 15 min. Supernatant were collected into new low-adsorption 1.5mL EP tubes. Then, 60 μL of Protein A beads for every 107 cells were washed in ChIP Dilution Buffer and resuspended in 50 μL Dilution Buffer per tube (100 μL per ChIP). Protein A beads were added to samples, which were rotated 1 h at 4 °C for precleaning. Sample supernatants were then separated using a magnet and transferred into new tubes, and then 7.5 μg antibodies per 107 cells were incubated at 4 °C overnight with rotation. The next day, 60 μL Protein A beads were washed and added to samples and then rotated 2 h at 4 °C. Beads were washed three times each with Low Salt Wash Buffer, High Salt Wash Buffer, and LiCl Wash Buffer. Chip-DNA was de-crosslinking with Proteinase K and purification using AmpureXP beads.For HiChIP:Briefly, 1.5×107 cells were crosslinked with 1% formaldehyde and lysed and then chromatin was digested using 400U MboI (NEB). Then 37.5μL biotin14-d-ATP was added and restriction-cut ends were ligated using 4000U T4 ligase (NEB). Pelleted nuclei were dissolved in nuclear lysis buffer (50 mM Tris-HCl, pH 7.5, 10 mM EDTA, 1% SDS, and protease inhibitors), sonicated and diluted in ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 7.5, and 167 mM NaCl). Immunoprecipitation was performed overnight at 4 °C by incubating RNA polⅡ antibodies (Biolegend) precoated on protein A-coated magnetic beads (Thermo Fisher Scientific). Immunocomplexes were washed three times each with low-salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 7.5, and 150 mM NaCl), high-salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 7.5, and 500 mM NaCl), and LiCl buffer (10 mM Tris-HCl, pH 7.5, 250 mM LiCl, 1% NP−40, 1% Na-Doc, and 1 mM EDTA), and beads were resuspended in DNA elution buffer (50 mM NaHCO3 and 1% SDS). After elution, ChIP samples were incubated with 10 mg/mL proteinase K 4 h at 55 °C, and then DNA was purified using AMPure XP Beads (Beckman). For RNA-seq: Total RNA was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext UltraTM RNA Library Prep Kit for Illumina (NEB, USA) following manufacturer's recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer(5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 250~300 bp in length, the library fragments were purified with AMPure XP system. Then 3 µl USER Enzyme (NEB, USA) was used with size selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95 °C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia) according to the manufacturer's instructions. After cluster generation, the library preparations were sequenced on an Illumina Novaseq 6000 platform and 150 bp paired-end reads were generated.For ChIP-Seq:Chip-seq library preparation was performed using eluted ChIP DNA with NEBNext Ultra DNA Library Prep Kit, according to manufacturer's protocol.For HiChIP:Streptavidin C1 beads were used to capture biotinylated DNA. QIAseq FX DNA Library Kits were used to generate the sequencing library. HiChIP libraries were size-selected to 300–700 bp using AMPure XP beads (Beckman) and subjected to 2 × 150-bp paired-end sequencing on HiSeq XTen (Illumina). Two biological replicates were analyzed for each experimental condition.