GSM8268768: Myobacterium tuberculosis, INH, 4 hrs, Biological Replicate 2; Mycobacterium tuberculosis; RNA-Seq
Drug-treated cultures and untreated controls were then harvested by centrifugation at timepoints 4, and 24 hours post drug treatment. To halt transcriptional responses during the RNA extraction process, GTC buffer was added to the cultures and they were washed twice in GTC buffer. The resulting pellets were stored in Eppendorf tubes containing ~1ml of GTC buffer (4M guanidinium thiocyanate) + 0.5% sarcosyl, 25 mM sodium citrate and 0.1M beta-mercaptoethanol. Samples were stored in aerosol tight O-ring caps at -80°C until RNA extractions were performed. On the day of extraction, the frozen GTC treated pellets were thawed at 37 degrees, harvested by centrifugation, resuspended in prewarmed triazole and transferred to 2ml O-ring tubes containing sterile zirconia beads. The triazole mixture was then bead beaten twice followed by the addition of chloroform and vigorous shaking for one minute. The samples were then centrifuged, and the aqueous layer was removed and placed in Qiagen RNeasy columns for RNA cleanup. Genomic DNA was removed from all samples through a DNAse digest on the columns prior to elution in pre-warmed RNase free water. The resulting RNA was then removed from the BSL-3 facility. Concentration and quality of RNA (260nm/280nm absorbance) was assessed using a nanodrop spectrophotometer and stored at -80°C until sample submission. Samples were submitted to the Broad Institute MOCP for sequencing, where they performed QC, library preparation, ribodepletion and paired-end sequencing on the Illumina NovaSeq 6000 following a modified version of the RNAtag-Seq method.