title: GSM8218487: ΔB11 + pPb11-B11 R1; Mycobacterium marinum; RNA-Seq
library_construction_protocol: Bacterial cultures (4 mL) were mixed with 0.2 volumes of stop solution (95% ethanol and 5% phenol) and immediately frozen in liquid nitrogen. Total RNA was isolated using the Trizol method, wherein cells were resuspended in 1 mL Trizol (Invitrogen) and 0.2 mL 0.1mm Zirconium/Silica beads (Biospec) for lysis. Bacterial lysis was achieved using a Precellys 24 homogenizer (4500 rpm, 3 cycles of 30 seconds, separated by 5-minute intervals on ice). After addition of 400 μL chloroform and centrifugation in a Phase Lock Gel tube (TIANGEN Biotech) at 13,000 rpm for 15 min, 500 μL of the aqueous phase was mixed with 450 μL isopropanol and precipitated at room temperature for 30 min. The RNA pellets were then washed with 80% ethanol, dissolved in ultra-pure water (Thermo Fisher Scientific), and the RNA concentration was determined using a NanoDrop 2000 (Thermo Fisher Scientific). Ribosomal RNAs was removed by Biotin-labeled rRNA probe. RNA was fragmented with BGI fragmentation buffer and was reverse transcribed with random hexamer. Double chain cDNA was then blunted and phosphorylated for the 5' end, and overhang A base in the 3' end and ligated with T-overhang adapter. After PCR application, samples were sequenced in BGISeq-500 platform