title: GSM4141807: P5m; Mus musculus; RNA-Seq
library_construction_protocol: Tumor tissue was isolated and single cell suspensions were prepared. Monocytes were FACS-sorted (CD45+CD11c-CD11b+Ly6g-Ly6c+) and RNA was isolated using RNeasy Plus Microkit (Qiagen). Complete cDNA was synthesized from 5 ng total RNA using the SmartScribe reverse transcriptase (Takara Bio) with a universally tailed poly-dT primer and a template switching oligo followed by amplification for 12 cycles with with the Advantage 2 DNA Polymerase (Takara Bio). After ultrasonic shearing (Covaris LE220), amplified cDNA samples were subjected to standard Illumina fragment library preparation using the NEBnext Ultra DNA library preparation chemistry (New England Biolabs). In brief, cDNA fragments were end-repaired, A-tailed and ligated to indexed Illumina Truseq adapters. Resulting libraries were PCR-amplified for 15 cycles using universal primers, purified using XP beads (Beckman Coulter) and then quantified with the Fragment Analyzer.