alias: GSM2212301
title: GSM2212301: Sample101.WT.BMDM.RNA-seq.PolyIC.6h; Mus musculus; RNA-Seq
library_construction_protocol: RNA-Seq: Total RNA was isolated using TRIzol LS (ThermoFisher Scientific) and resuspended  with UltraPure water (ThermoFisher Scientific) supplemented with 1U/μL SUPERase-In   (Ambion) then treated with TURBO DNA-free kit (Ambion). Poly(A) selection was performed  using the MicroPoly(A)Purist kit (Invitrogen) according to the manufacturer’s instructions.  Poly(A) RNA was fragmented using RNA Fragmentation Reagents (Ambion) for 10 min at 70°C  and purified by running through a Micro Bio-Spin P-30 column (Bio-Rad) according to the  manufacturer’s instructions. 30ng RNA was utilized for subsequent library preparation. ChIP-Seq:Briefly, for cFos, p65, Nrf2, and Smad3 ChIPs, macrophages were first cross-linked in 2mM dissuccinimidyl glutarate (Pierce 20593) in PBS for 30 minutes, followed by subsequent 1% formaldehyde (Sigma) cross-linking in PBS for 10 minutes at room temperature. For H3K27ac ChIPs, cells were cross-linked using 1% formaldehyde in PBS for 10 minutes at room temperature. After cross-linking, glycine (Sigma) was added to a final concentration of 0.2625 M to quench the reaction. Subsequently, cross-linked macrophages were centrifuged (5 minutes, 1200 rpm, 4°C), washed twice with PBS, and pellets snap frozen and stored at -80°C. For ChIP of H3K27Ac, p65, and Smad3, frozen cell pellets were resuspended in cell lysis buffer (10 mM HEPES/KOH pH 7.9, 85 mM KCl, 1 mM EDTA, 1.0% IGEPAL CA-630 (Sigma), 1x protease inhibitor cocktail (Roche), 1 mM PMSF). After five minutes lysis on ice, cells were centrifuged (5 minutes, 4000 rpm, 4°C), and the supernatant was removed. The pellet was then resuspended in nuclear lysis buffer (10 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na-Deoxycholate, 0.5% N-lauroylsarcosine, 1x protease inhibitor cocktail (Roche), and 1 mM PMSF) and the chromatin was sheared by sonication on wet ice with a Bioruptor Standard Sonicator (Diagenode) for three 15 min cycles each alternating 30 sec on and 30 sec off on the high setting. Additional Triton X-100 was added to the sonicated chromatin to 10% of the final volume and the lysate was cleared by centrifugation (5 minutes, 14000 rpm, 4˚C). Input was then saved for subsequent analysis. For cFos ChIP, pellets were suspended in 50 mM Tris pH 8.0, 60 mM KCl, 0.5% IGEPAL, 1x protease inhibitor cocktail (Roche), and 1 mM PMSF, followed by 10 minutes of incubation on ice and centrifugation at 2,000 x g for 3 minutes at 4°C. The pellet was then suspended in 0.5% SDS, 10 mM EDTA, 0.5 mM EGTA, 50 mM Tris pH 8.0, 1x protease inhibitor cocktail (Roche), and 1 mM PMSF. The chromatin suspension was sheared by sonication on wet ice with a Bioruptor Standard Sonicator (Diagenode) for three 15 min cycles each alternating 30 sec on and 30 sec off on the high setting, followed by centrifugation for 10 minutes at maximum speed at 4°C. The chromatin was diluted 5x with 1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris pH 8.0, 1x protease inhibitor cocktail (Roche), and 1 mM PMSF. An input sample was saved for subsequent analysis. ChIP-Seq: Antibodies against p65 (sc-372), cFos (sc-7202) and Nrf2 (sc-13032) were purchased from Santa Cruz Biotechnology, against Smad3 (ab28379) from Abcam, and against H3K27ac (39135) from Activ Motif. Protein A or G dynabeads (Invitrogen) pre-bound with antibody was added to the diluted cell lysate overnight at 4˚C. Immunoprecipitated complexes were washed three times with 20 mM Tris/HCl pH 7.4, 150 mM NaCl, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, three times with 10 mM Tris/HCl pH 7.4 at 20°C, 250 mM LiCl, 1% Triton X-100, 1% sodium deoxycholate, 1 mM EDTA, and two times with Tris-EDTA plus 0.1% Tween-20 before eluting two times with 50 µL elution buffer (TE, 1% SDS, 30 and 10 minutes, room temperature). Elution buffer was also added to the input. After pooling the eluted samples, the sodium concentration was adjusted to 300 mM and cross-links were reversed overnight at 65°C. Samples were treated with 0.5 mg/ml proteinase K for 1 hour at 55˚C and 0.25 mg/ml RNase A for 1 hour at 37˚C before DNA was isolated using the ChIP DNA Clean and Concentrator (Zymo Research) according to the manufacturer’s instructions. For library preparation, NEXTflex DNA barcode adaptors (BioO Scientific) were ligated to the genomic DNA. Polymerase chain reaction mediated library amplification was performed and final libraries were size selected on 10% TBE gels (Invitrogen). Libraries were PCR-amplified for 12-14 cycles, size selected by gel extraction and sequenced on a Hi-Seq 2000 (Illumina) for 51 cycles.
identifiers.namespace: GEO
identifiers.id: GSM2212301