GSM5001895: Jurkat_Input; Homo sapiens; ChIP-Seq
ChIP was performed using anti-H3K4me3 (C15410003; Diagenode). 5x106 Jurkat cells were used for each treatment with PBIT (10 uM) or OICR-9429 (25 uM) for 5 hours. Briefly, cells were cross-linked with 1% formaldehyde for 10 min at room temperature before the reaction was quenched with 250 mM glycine. Cell pellets were washed twice with PBS and nuclear lysates sonicated for 6 cycles using a Bioruptor Pico (Diagenode). Immunoprecipitation was performed with 5x105 Jurkat cells, complemented with 10% of Drosophila chromatin from S2 cells as spike in control (Orlando et al. 2014). Antibody (1 ug) was added to each sample and immunoprecipitation performed overnight on a rotator at 4 °C. 20 uL of magnetic beads (Dynabeads protein G, Novex Life technologies) was added to each sample. After 5 washes with three different buffers according to the BLUEPRINT protocol (http://dcc.blueprint-epigenome.eu/#/md/methods). ChIP-bound fractions were extracted by incubation for 20 min at room temperature with elution buffer (1% SDS, 0.1M NaHCO3). Cross-linking was then reversed by incubation at 65°C. Proteinase K (10mg/mL) was added to eliminate protein from the samples. DNA was extracted using the QIAquick MinElute PCR purification kit (Qiagen) following manufacturer's instructions. ChIP-Seq libraries were generated with the MicroPlex Library Preparation Kit (Diagenode), according to the manufacturer instructions. The libraries were sequenced in paired-end 50+30 bp mode using the NextSeq® 500/550 (Illumina) according to manufacturer's instructions.