Instrument: Illumina HiSeq 2000
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: SINGLE
Construction protocol: RNA Sequencing (RNA-seq) was performed and analyzed as described previously. Total RNA was prepared from approximately 1 million cells by using TRIzol or mirVana miRNA Isolation Kit (Thermo Fisher Scientific Inc.). 200 ng of total RNA was subsequently used to prepare RNA-seq library by using TruSeq SR RNA sample prep kit (FC-122-1001, Illumina) by following manufacturer’s protocol. RNA-seq: 200 ng of total RNA was subsequently used to prepare RNA-seq library by using TruSeq SR RNA sample prep kit (FC-122-1001, Illumina) by following manufacturer’s protocol. RNA-seq: The libraries were sequenced for 50 cycles (single read) with a HiSeq 2000 or HiSeq 2500 (Illumina) ChIP-seq: Cells cultured as indicated were cross-linked for 10 minutes with 1% formaldehyde and harvested. Cells were lysed by sonication and immunoprecipitated with anti-T-bet (sc-21003, Santa Cruz Biotechnology), anti- STAT1 (sc-592, Santa Cruz Biotechnology), anti-STAT2 (ab124283, AbCam), anti-H3K4m1 (ab8895, AbCam) and anti-H3K27ac (ab4729, AbCam) ChIP-seq: Libraries were prepared according to Illumina's instructions. Briefly, DNA was end-repaired and the blunt-ended, then phosphorylated ends were treated with Taq polymerase and dATP to yield a protruding 3- 'A' base for ligation of NEBNext adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation, DNA was PCR amplified with NEBNext index primers for 15 cycles and library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel, and quantitated by Qubit (Invitrogen). ChIP-Seq (size fractionation). The purified libraries were multiplexed and captured on an Illumina flow cell for cluster generation. Libraries were sequenced for 50 single read cycles on HiSeq 2000 or 2500 following the manufacturer's protocols.