GSM5673190: ΔaltRP/c ZLM1; Mycolicibacterium smegmatis; RNA-Seq
GSM5673190
Bacterial cell pellets in TRIzolTM were lysed via beating 3 times for 45 seconds at 7000 rpm in a MagNA Lyser (Roche) with cooling on ice for 3 minutes between cycles. Screw cap tubes were spun at 12,000xg for 5 minutes at 4°C and the supernatant was transferred to new 1.5 mL tubes. RNA and proteins were extracted from TRIzolTM supernatants concurrently, following the manufacturer's protocol. Precipitated RNA from the aqueous TRIzolTM fraction was resuspended in nuclease free water and purified using High Pure RNA Isolation kit (Roche, Cat. #: 11828665001) with omission of the on-column DNase digest step. Purified RNA was quantified using the ratio of absorbance at 260 and 280 nm using a NanodropTM (Thermo ScientificTM) spectrophotometer and digested in solution twice with TURBOTM DNase (InvitrogenTM, Cat. #: AM2239) following the manufacturer's directions. DNase-digested RNA was re-purified using the same kit as before. Ribosomal RNA was depleted from the samples using Ribo-Zero plus rRNA depletion kit (Illumina®, Cat. #:20037135) before being sequenced on an Illumina® MiSeq platform for generation of 150 base pair paired end reads. RNA sequencing (RNAseq) was performed by GENEWIZ® (South Plainfield, NJ 07080). Ribosomal RNA was depleted from the samples using Ribo-Zero plus rRNA depletion kit (Illumina®, Cat. #:20037135) before being sequenced on an Illumina® MiSeq platform for generation of 150 base pair paired end reads. RNA sequencing (RNAseq) was performed by GENEWIZ® (South Plainfield, NJ 07080). GENEWIZ prepared the libraries using Illumina's protocols for MISeq.