GSM1961539: Mus musculus T-bet-KO-Th1 RA H3; Mus musculus; ChIP-Seq
Cells were crosslinked by the addition of one-tenth volume of fresh 11% formaldehyde solution for 20 minutes at room temperature before the reaction was quenched by addition of glycine. Cells were rinsed twice with 1xPBS and flash frozen in liquid nitrogen. Cells were lysed with non-ionic detergent, nuclei washed and then lysed with ionic detergent. For RNA pol II ChIP, an alternative set of lysis and wash buffers were used (Rahl et al., 2010). Cells were sonicated on ice to solubilize and shear crosslinked DNA (24W for 10 x 30 second pulses using a Misonix Sonicator 3000). The resulting whole cell extract was cleared by centrifugation and then incubated overnight at 4°C with 100 µl of Dynal Protein G magnetic beads that had been pre-incubated with 10 µg of purified antibody or, for the case of T-bet, 10 µl of purified serum (see Table S6). 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 occasional vortexing and crosslinks then reversed in IP and input DNA by overnight incubation at 65°C. IP and input DNA were then purified by treatment with RNase A, proteinase K and phenol:chloroform extraction followed by ethanol precipitation.    H3K4me3 ChIP was performed on native chromatin. Chromatin was prepared by using the protocol of Feil and colleagues (http://www.epigenome-noe.net/researchtools/protocol.php?protid=2) with some minor modifications. Mono and dinucleosomal chromatin was recovered from nuclei treated with micrococcal nuclease (10U/µl for 7 mins) and chromatin quality assessed by agarose gel elecrophoresis and semi-quantitated using a nanodrop. ChIP for H3K4me3 and total H3 was performed with Protein G beads (Active Motif). DNA was purified by phenol/chloroform phase separation, ethanol precipitation, followed by clean up with Qiagen PCR purification columns. Libraries were constructed from ChIP and input DNA by standard Illumina protocols, except that DNA in the range 150-350bp was gel-purified after PCR-amplification. The libraries were quantified using a Qubit and Agilent bioanalyzer, pooled and subjected to 35 or 50 bp single-end read sequencing with an Illumina GAIIx or HiSeq 2500 sequencer.
GEO Accession
GSM1961539