GSM5902989: WT_U3; Mycobacterium tuberculosis H37Rv; RNA-Seq
25 mL GTC buffer (5 M guanidium thiocyanate, 0.5% sarcosyl, 0.5% Tween-80, 1% β mercaptoethanol (freshly added)) was added to 10 mL of bacterial culture, mixed, and cells were immediately harvested. Total RNA was extracted using MP Biomedicals™ FastRNA™ Pro Blue Kit (MP Biomedical, cat. No.- 116025050) and QIAGEN Rneasy Kit (Cat. No.- 74136)24. DNA contamination was removed from the isolated RNA using a TURBO DNA-free Kit (Invitrogen, Cat. No. -AM1907). Ribosomal RNA (16s and 23s rRNA) was depleted from these RNA samples using MICROBExpress Bacterial mRNA Enrichment Kit (Life Technologies, cat. No.- AM1905). rRNA depleted RNA (≈ 15 ng per sample) was fragmented and then used to prepare library using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs, Cat. No.-E7765s) and NEBNext Multiplex oligos for Illumina (New England Biolabs, Cat. No.-E7335s), according to the manufacturer's protocol. RNA libraries were sequenced using standard Illumina protocols