GSM9589794: Bleo Male 2; Mus musculus; miRNA-Seq
Paired mRNA and miRNA sequencing utilized services offered by LC Sciences (Houston, TX, USA).  Total lung RNA was extracted from the BW-matched cohort only (five sham-male, five sham-female, five bleomycin-male, five bleomycin-female) with TRIzol (Invitrogen) and randomized for downstream processing. Integrity was verified on a Bioanalyzer 2100 (Agilent); every sample showed RIN ≥ 7.0. Two complementary libraries were prepared from the same RNA aliquot for each animal. miRNA libraries were built from the remaining 1 µg RNA using the TruSeq Small-RNA kit (Illumina). Size-selected libraries (18–26 nt inserts) were single-end-sequenced (50 bp) on an Illumina HiSeq 2500, producing a median 9.2 million raw reads per sample. Filtering (adapter, low-quality, rRNA/tRNA/snoRNA) and mapping to miRBase v22 rat precursors were performed with ACGT101-miR v2023.1, allowing one mismatch in the mature region. Unmapped reads were BLASTed to Rnor_6.0; hairpins that met LC Sciences structural criteria (≥16 stem pairs, ΔG ≤ −15 kcal mol⁻¹, loop ≤ 20 nt) were annotated as novel miRNAs. Normalization used the median-ratio (DESeq) method; edgeR identified differentially expressed miRNAs (p < 0.05, |log₂ FC| ≥ 1). Putative targets were predicted by the intersection of miRanda and TargetScan-Rat