title: GSM5700426: D_Input_4h_LipidA_dBRD9_ChIPseq; Mus musculus; ChIP-Seq
library_construction_protocol: RNA was isolated using Quick-RNA Miniprep Kit (Zymo Research). For ChIP-Seq, 15 million BMDMs were collected and cross-linked first in 3mM disuccinimidyl glutarate (DSG) then in 1% formaldehyde. After quenching the excess formaldehyde with 125 mM glycine, the fixed cells were washed, pelleted and flash-frozen. Upon thawing, the cells were resuspended in lysis solution (50 mM HEPES-KOH pH 8, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP40, 0.25% Triton X-100 and incubated on ice for ten minutes. The isolated nuclei were washed with wash solution (10 mM Tris-HCl pH 8, 1 mM EDTA, 0.5 mM EGTA, 200 mM NaCl) and shearing buffer (0.1% SDS, 1 mM EDTA, 10 mM Tris-HCl pH 8) then sheared in a Covaris E229 sonicator for 10 minutes to generate DNA fragments between ~ 200-1000 bp. After clarification of insoluble material by centrifugation, the chromatin was immunoprecipitated overnight at 4C with antibodies against BRD9, BRD4, IRF9, STAT1, or STAT2. The next day, the antibody bound DNA was incubated with Protein A+G Dynabeads (Invitrogen) in ChIP buffer (50 mM HEPES-KOH pH 7.5, 300 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% sodium dodecyl sulfate). Antibody bound DNA were washed and treated with Proteinase K and RNase A and the purified ChIP DNA was used for library generation for subsequent sequencing. ChIP-Seq libraries were generated using the NuGen Ovation Ultralow Library System V2 following the manufacturer's instructions. RNA-Seq libraries were prepared using either Illumina TruSeq RNA Library Prep Kit v2 or Illumina TruSeq Stranded mRNA Kit following the manufacturer's instructions. ChIP-Seq and strand-specific polyA+ RNA-seq