GSM9600254: Bleo Male 1 RNA; Mus musculus; RNA-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. mRNA (poly-A) libraries were generated with the TruSeq-Stranded mRNA kit (Illumina). Poly-A RNA was captured with two rounds of oligo-dT magnetic-bead purification, chemically fragmented, converted to cDNA, end-repaired, A-tailed, ligated to indexed adapters, and amplified (15 cycles). Paired-end 150-bp reads were produced on an Illumina NovaSeq 6000 at LC Sciences (Houston, TX, USA), yielding a median of 7.9 G bases per sample with ≥97 % valid reads and Q30 > 98 %. Adapters and low-quality bases were removed with Cutadapt 1.10; quality was confirmed with FastQC 0.10.1. Clean reads were aligned to the Rattus norvegicus genome (Ensembl v112, Rnor_6.0) using HISAT2 2.0. Transcript assembly and gene-level quantification (FPKM) were performed with StringTie 1.3.4; counts were imported into DESeq2 and edgeR for differential expression. Genes with |log₂ fold-change| ≥ 1 and FDR < 0.05 were considered significant. Downstream GO and KEGG enrichment used clusterProfiler with Benjamini–Hochberg correction.