title: GSM8596725: TAX-R2; Mus musculus; RNA-Seq
library_construction_protocol: The monolayers were washed with PBS and macrophages lysed in Trizol reagent.  Mtb was centrifuged, the supernatant containing host RNA was removed, and the Mtb pellet was resuspended in fresh Trizol and 0.1 mm zirconia/silica beads. Mtb was mechanically disrupted with the Mini Bead-Beater Plus (Biospec Products). 70% of the sample containing host RNA was pooled with the sample containing Mtb RNA, the samples were treated with chloroform, and RNA was purified with the Trizol Plus RNA purification kit (Ambion). Purified total RNA was treated with DNAseI (ThermoFisher) and dried by rotary evaporation in RNA stabilization tubes (Azenta Life Sciences). Sample QC, dual rRNA depletion for bacteria and mouse, library preparation, Illumina sequencing (2x150 bp; 30M reads per sample), and differential gene expression analysis were performed by Azenta Life Sciences US, Inc. ERCC RNA Spike-In Mix kit (cat. 4456740) from ThermoFisher Scientific, was added to normalized total RNA prior to library preparation following manufacturer's protocol. rRNA depletion was performed using QIAGEN FastSelect rRNA Bacteria + HMR Kit or HMR/Bacteria (Qiagen, Germantown, MD, USA), which was conducted following the manufacturer's protocol. RNA sequencing libraries were constructed with the NEBNext Ultra II RNA Library Preparation Kit for Illumina by following the manufacturer's recommendations. Briefly, enriched RNAs are fragmented for 15 minutes at 94 °C. First strand and second strand cDNA are subsequently synthesized. cDNA fragments are end repaired and adenylated at 3'ends, and universal adapters are ligated to cDNA fragments, followed by index addition and library enrichment with limited cycle PCR. Sequencing libraries were validated using the Agilent Tapestation 4200 (Agilent Technologies, Palo Alto, CA, USA), and quantified using Qubit 2.0 Fluorometer (ThermoFisher Scientific, Waltham, MA, USA) as well as by quantitative PCR (KAPA Biosystems, Wilmington, MA, USA).