GSM8854264: macrophages present in the steady state, rep 1; Mus musculus; RNA-Seq
Total RNA was extracted, from 3 biological replicates per condition, using the RNAqueous-Micro Kit (Invitrogen), according to the manufacturer's instructions. RNA samples were quantified and checked for integrity using an Agilent 2100 Bioanalyzer (Agilent Technologies). 30 ng of total RNA was used to generate barcoded RNA-seq libraries using the NEBNext Ultra II Directional RNA Library preparation kit (New England Biolabs) according to manufacturer's instructions. First, poly A+ RNA was purified using poly-T oligo- attached magnetic beads followed by fragmentation and first and second cDNA strand synthesis. Next, cDNA ends were repaired and adenylated. The NEBNext adaptor was then ligated followed by second strand removal, uracile excision from the adaptor and PCR amplification. The size of the libraries was checked using the Agilent 2100 Bioanalyzer and the concentration was determined using the Qubit® fluorometer (ThermoFisher). Libraries were sequenced at a final concentration of 650 pM using a P2 flow cell in a NextSeq 2000 sequencer (Illumina) to generate 100pb single reads.