title: RNA seq of macaca mullata: lung
design: Lung tissue pieces from macaques were weighed for genomic DNa extraction, transferred to a petri dish and tissue dissociation was performed using a tissue homogenizer. Chelex method was used for Mtb DNA extraction from macaque lung tissue. Post dissociation, the lung samples were resuspended in 2 mL tubes containing lysing matrix B and chelex. The bacteria was lysed mechanically using a bead beater for 30s at 3 800 rpms for 3 cycles. The tubes were transferred to a heating block and boiled at 100 degrees Celsius for 15 mins. This was followed by water bath sonication for 15 mins. The prepared samples were then used for stranded mRNA-Seq library prep, and 75 bp PE sequencing on Illumina NextSeq 500 Mid Output Platform. RNA quality control, library Agilent quality control was also performed. Qiagens Ingenuity Pathway Analysis (IPA) software was used to analyze the RNA-seq data to identify significant pathways.  Raw, demultiplexed .fastq sequence files were uploaded to Partek Flow analysis software for standard RNA-seq analysis, including differential gene expression analysis.  Briefly, bases with a Phred score less than Q30 were trimmed, aligned to RefSeq Mmul10 reference assembly (GCF_003339765.1) with STAR aligner v2.7.8a using default settings, and quantified using the Quantify to annotation model (Partek E/M) tool with the RefSeq Mmul10 annotation release as the transcript model.  Raw gene counts were normalized by median ratio (transformed on samples), and subsequent DESeq2 differential expression analysis was carried out by comparing cART treated vs. cART+3HP treated samples with the ART+3HP treated samples as reference levels.