GSM5018699: WT_Day10_Rep3_RNAseq; Homo sapiens; RNA-Seq
ChIP-seq: Cells were dissociated with Accutase and washed with PBS. Cells were cross-linked with 2 mM Di(N-succinimidyl) glutarate in PBS for 45 min at room temperature with shaking and then with 1% methanol-free paraformaldehyde for 12.5 min at room temperature with shaking. Fixation was quenched with 0.125 M Glycine for 5 min at room temperature with shaking. Chromatin was fragmented by sonication on a Microson ultrasonic cell disruptor XL Misonix wand sonicator with output setting of 10W using 30 or 34 sonication cycles (15s on, 30s off) for primed and naïve cell chromatin respectively. Fragmented chromatin was incubated with 10 µg of antibody overnight at 4C. Chromatin-antibodies complexes were isolated using Magnetic Protein A or G dynabeads and washed. Eluted chromatin was de-crosslinked and DNA was purified using AMPure XP beads. RNA-seq: Total RNA was extracted in TRIzol Reagent. ChIP-Seq libraries were prepared using the NEBNext Ultra II DNA library preparation kit for Illumina using the manufacturer's protocol, with libraries indexed using NEBNext Multiplex Oligos for Illumina (Index Primers Set 1 and Set 2). Library fragment size and concentration were determined using the Qubit fluorometer double stranded DNA high sensitivity assay kit, using an Agilent Bioanalyzer 2100 and by KAPA Library Quantification Kit. Samples were sequenced on an Illumina NextSeq500 instrument as HighOutput 75 bp single-end reads at the Babraham Institute Next Generation Sequencing Facility. RNA-seq: Indexed libraries were constructed from 500ng total RNA using the NEBNext Ultra RNA Library Prep Kit for Illumina with the Poly(A) mRNA Magnetic Isolation Module. Library fragment size and concentration was determined using an Agilent Bioanalyzer 2100 and KAPA Library Quantification Kit.