GSM9268237: ChIP-Seq, H3K4me1, Macrophage, STAT1alpha KI, Untreated, 14h, rep2; Mus musculus; ChIP-Seq
Cells were crosslinked with 1 % formaldehyde (ThermoFisher Scientific, 28908) in PBS for 10 minutes (min) at room temperature (RT) on a shaker. Cells were quenched with 0.125 M glycine for 20 min at RT. Cells were harvested and washed twice in ice-cold PBS containing 1x protease inhibitor tablets (PI, Sigma-Aldrich, S8820-2TAB) and 1 mM PMSF (Sigma-Aldrich, SLBX4318). Cells were centrifuged for 5 min at 300 × g at 4 °C, pellets were snap-frozen in liquid nitrogen and stored at -80 °C overnight. Pellets were thawed on ice for 30 min and resuspended in 1 mL of sonication buffer (10 mM Tris pH 8.0, 0.25% SDS, 2mM EDTA) supplemented with 1 mM PMSF and 1x PI. Samples were sonicated in 15 mL polypropylene tubes (Falcon, 352099) suitable for the Bioruptor® Pico (Diagenode) for 20 minutes. Sonicated samples were centrifuged for 10 min at 14000 × g at 4 °C and the supernatant was diluted in 1.5 mL equilibration buffer (10 mM Tris, 233 mM NaCl, 1.66 % TritonX-100, 0.166 % DOX, 1 mM EDTA, 1 mM PMSF, PI). 20% and 10% of total chromatin were used for each immunoprecipitation (IP) and input control, respectively. 1 mg of anti-H3K27ac (Diagenode, C15410196) or 2 mg of anti-H3K4me1 (Abcam, 8895) antibody was added to the sonicated chromatin aliquots. All samples were incubated on a rotator overnight at 4°C. Magnetic beads (ThermoFisher Scientific, 10004D) were washed twice in RIPA-LS buffer (10 mM Tris-HCL pH 8.0, 140 mM NaCl, 1mM EDTA pH 8.0, 0.1% SDS, 0.1% sodium deoxycholate, 1% triton X-100) containing 0.1% BSA. The beads were blocked overnight in RIPA-LS buffer containing 0.1% BSA at 4°C. Input control samples were reversed crosslinked overnight at 65°C. The next day 10 μl of the magnetic beads were added and samples were incubated 3 to 4 hours at 4°C (on a rotator). The beads were washed once with RIPA-LS buffer, RIPA-HS buffer (10 mM Tris-HCL pH 8.0, 500 mM NaCl, 1mM EDTA pH 8.0, 0.1% SDS, 0.1% sodium deoxycholate, 1% triton x-100), RIPA- LiCl buffer (10 mM Tris-HCL pH 8.0, 1mM EDTA pH 8.0, 0.5% sodium deoxycholate, 0.5 % NP-40, 250 mM LiCl) and TE buffer (10 mM Tris-HCL pH 8, 1 mM EDTA). The beads were gently resuspended in a 25 mL tagmentation reaction mix. The tagmentation reaction mix contained 5x tagmentation buffer (50 mM Tris pH 8, 0.25% SDS, 2 mM EDTA), 19 mL nuclease-free water and 1x tagment DNA enzyme (Illumina, 15027865). The reaction mix was incubated for 10 min at 37°C. The beads were washed once with RIPA-LS buffer and 10 mM Tris pH8 and then resuspended in 20 mM EDTA. Samples were heated for 30 min at 50°C and 25 mL NEB Next High fidelity 2x master mix (NEB, M0480S) was preheated for 20s at 98°. 10.5 mL 20 mM MgCl2 was added to the master mix and the mixture was added to the samples. The samples were incubated for 5 min at 72°C and 10 min at 95°C and cooled on ice and 2x forward and barcoded reverse primers were added. Primers used for the ChIPseq analysis are listed in Supplementary Table 6. An enrichment PCR was performed with the following program: 98°C for 30s, 15 cycles (98°C for 10s, 63°C for 30s and 72°C for 30s) and 72°C for 1 min. The supernatant was removed using DynamagTM-96 Side Magnet (ThermoFisher Scientific, 12331D) and stored at -80°C. DNA from the input samples were eluted using Qiagen MinElute Kit (Qiagen, 28006). DNA was quantified using Qubit Fluorometer (ThermoFisher Scientific). To 2 ng of input DNA, 12 mL of the tagmentation reaction mix (1 mL of 5X tagmentation buffer, 1 mL of 1:10 diluted Tagment enzyme and nuclease-free water) was added. The tagmented input DNA was purified with a Qiagen MinElute kit. 2x forward, barcoded reverse primers and 20 mL NEBNext High fidelity 2X master mix were added to the eluted input DNA. An enrichment PCR was performed with the abovementioned program, except the cycle number was 12. The libraries of the samples and input controls were purified and the size was selected by adding 2x AMPureXP beads (Beckman Coulter, A63880). The mixture was resuspended and incubated for 10 min at RT. The supernatant was discarded, and the beads were washed twice with 80% EtOH for 5 seconds and air dried for 5 min on the magnet. The beads were removed from the magnet to elute the DNA, and 50 mL of water was added for 10 min at RT. The beads were placed back on the magnet for 5 min, and the supernatant was removed into a new tube. DNA was measured on the Qubit Fluorometer. For quality check, the fragment size of the DNA was analyzed on Agilent High Sensitivity D1000 TapeStation. 4 nM of samples were pooled according to barcode and DNA fragment size. Library preparation was conducted on 1 ng of cDNA using the Nextera XT DNA Sample Preparation Kit (Illumina) followed by SPRI (Beckman Coulter) size selection. Sequencing was performed by the Bio-medical Sequencing Facility at CeMM using the Illumina HiSeq 3000/4000 platform and the 50 bp single end configuration.