title: Illumina HiSeq 2500 paired end sequencing; Hydrogen sulfide stimulates Mycobacterium tuberculosis respiration and growth to exacerbate TB disease
design: Hydrogen sulfide stimulates Mycobacterium tuberculosis respiration and growth to exacerbate TB disease
library_construction_protocol: RNA was isolated from actively growing mycobacterial cells (0.6-0.8 OD600) using Trizol according to established protocols previously reported (Saini V et al. 2016, Cell reports).  Briefly, cells were exposed to 25 µM GYY4137 for 1 hr under carefully controlled conditions (n=3/group) and RNA isolated.  Unexposed cells received spent GYY4137 ( without any capacity to produce Hydrogen sulfide).  M. tuberculosis H37Rv (obtained from BEI Resources (NR-123), was grown in Difco Middlebrook (MB), 7H9 (broth) supplemented with 0.2% glycerol, 1X ADS (albumin, dextrose, sodium chloride) and 0.02% tyloxapol at 37 °C. To prepare H2S donor compound, a 10 mM GYY4137 stock solution was prepared in water in a 5 ml tube and was immediately diluted to make a working stock and added to cultures to achieve the desired final concentration.  Experimental control samples received a time-spent 25 µM GYY4137 (referred to as spent GYY) that was previously aerated for at least 120 days. Mycobacterium tuberculosis H37Rv cells (treatment group) were exposed to 25 µM GYY4137 for 1 hr under carefully controlled conditions (n=3/group).  Unexposed or untreated cells received spent GYY4137, as a vehicle control which does not have any capacity to release hydrogen sulfide. Trizol method of RNA isolation Ribosomal RNA reduction was performed using a Ribo-Zero kit (Illumina, USA) and libraries were prepared using TruSeq library generation kits (Illumina USA).
attributes.tag: Experimental Factor: compound
attributes.value: GYY4137
attributes.tag: Experimental Factor: dose
attributes.value: 25