GSM1647841: RNASeq withIFNg; Mus musculus; RNA-Seq
For ChIP-Seq, chromatin from 1 × 108 cells was sonicated for 20 cycles of 12 sec on/48 sec off, into 200-500 bp fragments, DNA ends were repaired using polynucleotide kinase and Klenow enzyme, and treated with Taq polymerase to generate a protruding 3’ “A” nucleotide. DNA fragments of ~250-450 bp were purified from agarose gels and used for cluster generation and sequencing. Antibodies against IRF8 were used (Santa Cruz Biotechnology, Cat# sc-6058x). 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