title: GSM2974647: C57_BMDM_5GRO_KLA_1h_rep1; Mus musculus; OTHER
library_construction_protocol: Femur, tibia and iliac bones from the different mouse strains were flushed with DMEM high glucose (Corning) and red blood cells were lysed using red blood cell lysis buffer (eBioscience). After counting, 20 million bone marrow cells were seeded per 15cm non-tissue culture plates in DMEM high glucose (50%) with 20% FBS (Omega Biosciences), 30% L929-cell conditioned media (as source of M-CSF), 100 U/ml penicillin/streptomycin+L-glutamine (Gibco) and 2.5μg/ml Amphotericin B (HyClone). After 4 days of differentiation, 16.7 ng/ml mouse M-CSF (Shenandoah Biotechnology) was added to the media. After an additional 2 days of culture, non-adherent cells were washed off with room temperature DMEM high glucose and macrophages were obtained as a homogeneous population of adherent cells which were scraped and subsequently seeded onto tissue culture-treated petri dishes overnight in DMEM high glucose containing 10% FBS, 100 U/ml penicillin/streptomycin+L-glutamine, 2.5μg/ml Amphotericin B and 16.7 ng/ml M-CSF. For KLA activation, macrophages were treated with 10 ng/mL KLA (Avanti Polar Lipids) for 1 or 6 hours. GRO-seq and 5'GRO-seq: Nascent transcription was captured by global nuclear run-on sequencing (GRO-seq) (Core et al., 2008) and nascent transcription start sites by 5'GRO-seq (Lam et al., 2013). Nuclei were isolated from BMDMs using hypotonic lysis [10 mM Tris-HCl (pH 7.5), 2 mM MgCl2, 3 mM CaCl2; 0.1% IGEPAL CA-630] and flash frozen in GRO-freezing buffer [50 mM Tris-HCl (pH 7.8), 5 mM MgCl2, 40% Glycerol]. 3-5 x 106 BMDM nuclei were run-on with BrUTP-labelled NTPs as described (Duttke et al., 2015) with 3x NRO buffer [15mM Tris-Cl (pH 8.0), 7.5 mM MgCl2, 1.5 mM DTT, 450 mM KCl, 0.3 U/μL of SUPERase In, 1.5% Sarkosyl, 366 μM ATP, GTP (Roche), Br-UTP (Sigma Aldrich) and 1.2 μM CTP (Roche, to limit run-on length to ~40 nt)]. Reactions were stopped after five minutes by addition of 500 μL Trizol LS reagent (Invitrogen), vortexed for 5 minutes and RNA extracted and precipitated as described by the manufacturer.
attributes.tag: GEO Accession
attributes.value: GSM2974647