GSM1543812: CD4.WT.IL21.STAT3; Mus musculus; ChIP-Seq
For ChIP-Seq, Cells were fixed with 1% formaldehyde at 37°C for 10 min, and chromatin from 2.5 x 107 cells was sonicated for 20 cycles (15 sec on/45 sec off) to produce 250-450 bp fragments. STAT3-linked chromatin was immunoprecipitated using anti-STAT3 (13-7000, Invitrogen) antibody bound to Dynabeads.  The ends of isolated DNA were repaired using the DNA END-Repair kit (Epicentre Biotechnologies), and a 3’ “A” nucleotide was added with Taq polymerase.  Two mixed Solexa adaptors (Illumina) were ligated to DNA ends and primers complementary to these adaptors (PE 1.0 and 2.0, Illumina) were used to amplify the DNA for 18 cycles.  DNA fragments of 250-450 bp were purified from 2% E-gels and used for cluster generation and sequencing. For RNA-Seq,  Total RNA was isolated, and 5 μg per sample were used for mRNA purification using Dynal oligo(dT) beads (Invitrogen).  Double-stranded cDNA was synthesized with random hexamer primers, SuperScript II, DNA polymerase I, and T4 DNA polymerase (Invitrogen) and fragmented by Bioraptor.  After end repair (Epicentre DNA END-Repair kit) and the addition of an “A” nucleotide to the 3’ ends with Taq DNA polymerase, two mixed Solexa adaptors (Illumina) were ligated to DNA ends using T4 DNA ligase (New England Biolabs).  250-450 bp fragments were isolated on 2% E-Gels (Invitrogen) and amplified for 18 cycles using PE 1.0 and 2.0 primers (Illumina) and Phusion High Fidelity PCR Master Mix (New England Biolabs).  PCR products were run on a gel, and 250-450 bp fragments were purified PCR products were barcoded (indexed) and sequenced on Illumina HiSeq 2000 platform