GSM4594621: CDC_Rv1963c_D1_UT_U_2; Mycobacterium tuberculosis; RNA-Seq
RNA was isolated using published protocols (Rustad TR, Harrell MI, Liao R, Sherman DR (2008) The Enduring Hypoxic Response of Mycobacterium tuberculosis. PLoS ONE 3(1): e1502). In summary, frozen cell pellets Pellets were resuspended in Trizol, transferred to a tube containing Lysing Matrix B (QBiogene, Inc.), and vigorously shaken at max speed for 30 sec in a FastPrep 120 homogenizer (Qbiogene) three times, with cooling on ice between steps. This mixture was centrifuged at max speed for 1 min and the supernatant was transferred to a tube containing 300 mL chloroform and Heavy Phase Lock Gel (Eppendorf North America, Inc.), inverted for one minute, and centrifuged at max speed. The aqueous phase was then precipitated with 270 mL isopropanol and 270 mL high salt solution (0.8M Na citrate, 1.2M NaCl). RNA was cleaned using an RNeasy kit following manufacturer's recommendations (Qiagen). Messenger RNA was enriched using the RiboZero rRNA removal (bacteria) magnetic kit (Illumina Inc.) according to manufacturer's instructions. Libraries were prepared for Illumina sequencing using the NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs) according to manufacturer's instructions and using the AMPure XP reagent (Agencourt Bioscience Corporation) 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 associated with each replicate and multiplexing up to 48 libraries per sequencing run. The prepared libraries were quantified using the Kapa qPCR quantification kit, and were sequenced at the University of Washington Northwest Genomics Center with the Illumina NextSeq 500 High Output v2 Kit (Illumina Inc).
GEO Accession
GSM4594621