GSM5240582: CSC24h [dw_rna_seq_12]; Mycobacterium tuberculosis; RNA-Seq
Cultures (30 ml), were collected, resuspended in 1 ml RNAProBlue solution, transferred to a 2 ml tube containing 0.2 mm matrix B beads (MP Biomedicals) and frozen at -80 °C. After defrosting, bacteria were lysed by bead beating at 3 800 rpm for 3 cycles of 30 s each using a BioSpec Mini-Beadbeater-24 and cellular debris removed by centrifugation at 15 000´g for 15 minutes. Chloroform (300 μl) was added and the reaction vortexed for 10 seconds (s). The Nucleospin RNA kit (Machery-Nagel) kit was used for RNA extraction according to manufacturer's instructions.the RNA was also treated with Turbo DNase from the TURBO DNA-free Kit (Ambion) kit according to manufacturer's instructions. Following inactivation of the DNase using inactivation reagent (Ambion), the RNA Clean & Concentrator Kit (Zymo) was used to get rid of the DNase buffer and RNA was eluted in RNase-free water. The Zymo-Seq RiboFree Total RNA Library Kit was used for library preparation according to the manufacturer's instructions. RNA (500 ng) was used and ribosomal depletion done for 30 minutes, followed by 14 cycles of PCR. RNA-seq was done using the Illumina NextSeq 500 V2.5 kit according to the manufacturer's instructions on the NextSeq 500 machine by the UTHSCSA Genome Sequencing Facility.