GSM8862692: H37Rv_BTZ@64ng/mL+48h_b11_2; Mycobacterium tuberculosis; RNA-Seq
Bacterial pellets suspended in TRIzol were transferred to a tube containing Lysing Matrix B (QBiogene) and vigorously shaken in a homogenizer. The mixture was centrifuged, and RNA was extracted from the supernatant with chloroform, followed by RNA precipitation by isopropanol and high-salt solution (0.8 M Na citrate, 1.2 M NaCl). Total RNA was purified using a RNeasy kit following the manufacturer's recommendations (Qiagen). rRNA was depleted from samples using a custom-designed set of biotinylated oligonucleotides based on the method described in PMID: 32317317. Illumina sequencing libraries were prepared using the NEBNext Ultra II RNA Library Prep kit for Illumina (New England Biolabs, Ipswich, MA) according to the manufacturer's instructions, and using the AMPure XP reagent (Agencourt Bioscience Corporation, Beverly, MA) for size selection and cleanup of adaptor-ligated DNA. We used the NEBNext Multiplex Oligos for Illumina (Dual Index Primers Set 1) to barcode the libraries to enable sample multiplexing per sequencing run. The prepared libraries were quantified using the Kapa quantitative PCR (qPCR) quantification kit.