GSM2525690: Treated_24h_Rep1; Mycobacterium tuberculosis; RNA-Seq
Cells were resuspended in trizol and disrupted using Zirconia beads (Biospec, Bartlesville, OK, USA) by beating six times for 30 sec and cooling on ice for one min in between. Beads were removed and chloroform was added to trizol (0.2:1, v/v). Samples were vortexed and centrifuged to extract solubilized RNA in the aqueous phase. To precipitate RNA, molecular grade 100% ethanol (Sigma-Aldrich Aldrich, USA) was added to the aqueous phase and incubated at -80⁰C overnight. The RNA was pelleted by centrifugation (×17,000g) at 4⁰C, for 15 min followed by washing with 75% ethanol. DNA contamination was removed by treating with 10 µL DNase1 (New England Biomed, Ipswich, MA, USA) at 37⁰C for 30 min. Then 100 µL of acid phenol (Sigma-Aldrich, USA) was added to the samples and vortexed. After centrifugation (×17,000g) for one min, the aqueous phase was collected and transferred to clean RNAse-free tubes, followed by the addition of 33 µL sodium acetate (Sigma-Aldrich, USA) and 250 µL of 100% ethanol. Samples were gently mixed by inverting and placed at -80⁰C overnight. Samples were centrifuged (×17,000g) for 15 min at 4⁰C to collect RNA pellets. Pellets were further washed with 80% ethanol. After ethanol removal, pellets were air-dried at room temperature for five min and reconstituted in 15 µL of RNAse-free water (Corning, USA). Illumina Stranded Total RNA Library Prep Kit (Illumina, San Diego, CA, USA) with the Epicentre Ribo-Zero Gram positive bacteria ribosomal RNA depletion (Illumina, USA). The pool was loaded on one lane of a HiSeq Rapid Run flow cell (v1) and sequenced in a 1×50 bp single end (SE50) format using Rapid SBS reagents. Base calling was performed by Illumina Real Time Analysis (RTA, Illumina, USA) v1.18.61 and output of RTA was de-multiplexed and converted to FastQ format with Illumina Bcl2fastq v1.8.4 (Illumina, USA). To compensate for the low number of reads obtained for one sample, MiSeq (SE50) sequencing was performed with this one library. Reads from both the HiSeq and MiSeq runs were combined for that particular sample.