title: RNAseq of mus musculus macrophages
design: RNA was extracted from pellets of Peripheral blood macrophages (PBM), LM and FM. Total RNA was extracted using TRIzol Reagent. Total RNA of each sample was subsequently quantified and qualified by Agilent 2100 Bioanalyzer (Agilent Technologies) and NanoDrop (Thermo Fisher). 1 g total RNA with RIN value above 7 was allowed for following library preparation. The poly(A) mRNA isolation was done using NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB). The mRNA fragmentation and priming were done using NEBNext First Strand Synthesis Reaction Buffer and NEBNext Random Primers. First strand cDNA was synthesized using ProtoScript II Reverse Transcriptase and the second-strand cDNA was synthesized using Second Strand Synthesis Enzyme Mix. Size selection of Adaptor-ligated DNA was performed using AxyPrep Mag PCR Clean-up (Axygen), and fragments of ~360 bp were recovered. Each sample was amplified by PCR for 11 cycles using P5 and P7 primers. Then libraries with different index were multiplexed and loaded on an Illumina HiSeq platform in accordance with manufacturers instructions (Illumina).