GSM2346476: 14050-MM_WT_0h; Mus musculus; RNA-Seq
Mice were sacrificed via CO2 euthanasia. Femur and tibia bones were harvested and bone marrow flushed with DMEM. Collected cells pelleted and resuspended in 10mL of 1X Red blood Cell (RBC) Lysis buffer for 5min. Cells were resuspended in 20mL of fresh DMEM. 2.5e6 bone-marrow cells plated in a 15-cm TC dish in 20mL of BMDM Media (DMEM, 20% FBS, 30% L929 condition media, and 1% Pen/Strep) and grown at 5% CO2 and 37°C. BMDM media was completely replaced on day 3. On Day 6, BMDMs were replated at a concentration of 1e6 cells per well in a 6 well plate and incubated for 16hr. Stimulations and treatments began at day 7. Stimulated cells were lysed using an RNAeasy kit (Qiagen) with DNaseI digestion (Qiagen) as per manufacturer’s protocol. RNA-seq libraries were prepared from polyA+ selected RNA using the TruSeq RNA Sample Preparation kit at the Millard and Muriel Jacobs Genetics and Genomics Laboratory at Caltech. Libraries were sequenced on the Illumina HiSeq 2500 generating single-end 50 bp reads. TruSeq RNA Sample Preparation Kit