Mice were euthanized, alveolar macrophages (~100K cells) were isolated via FACS as described before (Misharin et al., AJRCMB, 2013; Misharin et al., JEM, 2017). Cells were immediately lysed in 350 ul of RLTplus buffer supplemented with BME. RNA was extracted using Qiagen RNeasy Plus protocol with genomic DNA removal step. All samples had RIN over 7 according to Agilent TapeStation 4200. RNA-seq libraries were prepared from 100 ng of total RNA, starting with poly(A) enrichment and followed by NEB Next RNA Ultra I chemistry. Libraries were quantified and assessed on Qubit fluorimeter (Invitrogen) and TapeStation 4200, correspondingly, multiplexed and sequenced on NextSeq 500 instrument (Illumina), using V2 high output reagent kit, 75 bp, single end reads, to the average sequencing depth of 6x10^6 reads per sample. Over 94% of reads had Q score over 30.
GEO Accession
GSM2610751