GSM5885624: EL4_GATA3_Dox_Tbet_DOX_04_Flag-Tbet_ChIP; Mus musculus; ChIP-Seq
Cells were lysed with non-ionic detergent, the nuclei washed and then lysed with ionic detergent. Cells were sonicated on ice to solubilize and shear crosslinked DNA (27W for 10x 30 second pulses using a Misonix Sonicator 3000). The resulting whole cell lysate was cleared by centrifugation and then incubated overnight at 4°C with 50 µl of Protein G magnetic Dynabeads that had been pre-incubated with 5 µg of purified antibody (anti-FLAG, M2 Sigma; anti-HA, 3F10 Roche; anti-H3K27ac, ab4729 Abcam). Beads were washed 6 times with RIPA buffer and 1 time with TE containing 50 mM NaCl. Bound complexes were eluted from the beads by heating at 65°C with rocking for 2 hrs and crosslinks then reversed in IP and input DNA by incubation at 65°C for 6 hrs. IP and input DNA were then purified by treatment with RNase A, proteinase K and isolated with KAPA Pure beads. Libraries were constructed from ChIP DNA by standard Illumina protocols, except that DNA in the range 150-350 bp was gel-purified after PCR-amplification. The libraries were quantified using a Qubit fluorometer and Agilent bioanalyzer, pooled and subjected to 75 bp single-end sequencing with a NextSeq 550 sequencer