GSM2759907: Jurkat input DNA (unary control); Homo sapiens; ChIP-Seq
ChIP was performed in duplicate for each TF using the ChIP-IT High Sensitivity Kit (Active Motif). Briefly, 15 x 10^6 cells were X-linked with formaldehyde (1.14%) for 15mins and nuclei extracted following the protocol. Chromatin was re-suspended in 500µl of ‘ChIP Buffer’ and fragmented to 200-800bp using the BioRuptor Plus (Diagenode) for 6mins (30sec ON, 30sec OFF, HIGH mode). Per ChIP, 150µl of chromatin was incubated overnight at 4°C with 4µg of antibody. Immune-complexes were captured and purified on Protein G agarose beads and washed using the solutions provided. Reverse X-linking of ChIP’d DNA was performed by with RNase A (10µg) and incubation at 37°C for 30mins, then addition of Proteinase K (20µg) and incubation at 55°C for 30mins then 80°C for 2hours. Samples were purified on the Active Motif DNA purification columns. Quantification of DNA was performed with the Qubit HS ds-DNA assay (ThermoFisher). An aliquot of sonicated chromatin was used as input DNA control. Libraries were created from the purified fragments for both ChIP and input using the NEBNext ChIP-Seq Library Prep Master Mix Set for Illumina (NEB). Purification and size selection was performed using the AMPure XP Beads (Beckman Coulter). Quality assessment of each library was carried out using the Agilent 2100 Bioanalyzer and the Agilent High Sensitivity DNA Kit. Libraries were sequenced on the Illumina HiSeq 2500 platform with single-end 50bp reads.
GEO Accession
GSM2759907