RNA-Seq analysis of Wt mouse BMDM cells differentiated in the presence of MCSF (24hIL4_3hLPS_rep2)
RNA-Seq libraries were prepared from at least three biological replicates by using TruSeq RNA Sample Preparation Kit (Illumina) according to manufacturer protocol. Briefly, 2.5_g total RNA was used for the library preparation. In the first step poly-A tailed RNA molecules (mRNA) were purified with poly-T oligo-attached magnetic beads. Following the purification mRNA is fragmented using divalent cations at 85_C, then first strand cDNA was generated using random primers and SuperScript II reverse transcriptase (Invitrogen, Life Technologies). This was followed by the second strand cDNA synthesis, then double stranded cDNA fragments went through an end repair process, the addition of a single ?A_ base and then barcode indexed adapter ligation. Adapter-ligated products were enriched with adapter specific PCR to create cDNA library. Agarose gel electrophoresis was performed on E-Gel EX 2% agarose gel (Invitrogen, Life Technologies) and the library was purified from the gel using QIAquick Gel Extraction Kit (Qiagen). Fragment size and molar concentration were checked on Agilent BioAnalyzer using DNA1000 chip (Agilent Technologies). Libraries were sequenced with Illumina HiSeq 2500 sequencer.