title: GSM2974782: NOD_BMDM_RNA_polyA_KLA_1h_rep1; Mus musculus; RNA-Seq
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. RNA-Seq: Total RNA was isolated from cells and purified using Quick RNA mini prep columns and RNase- free DNase digestion according to the manufacturer's instructions (Zymo Research). RNA-seq libraries were prepared from poly(A)-enriched mRNA, as previously described (Oishi et al., 2017). Libraries were sequenced on HiSeq 4000 or NextSeq 500.
attributes.tag: GEO Accession
attributes.value: GSM2974782