GSM4518463: ΔB11 + pSMT3 (Mock)-R2; Mycobacterium marinum M; RNA-Seq
Bacterial cultures at OD600~2.0 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. Briefly, 4.0 OD600 of cells were resuspended with 1mL Trizol (#15596018, Invitrogen) and 0.2 mL 0.1mm Zirconia/Silica beads (#11079101z, Biospec).   Bacteria cells were lysed with a Precellys 24 homogenizer (4500 rpm, 3x30s, separated by 5min intervals on ice,). After mixing with 400 μl chloroform and centrifugation in a Phase Lock Gel tube (#WM5-2302820, TIANGEN Biotech) at 13,000 rpm for 15 min, 500 ul of the aqueous phase was mixed with 450 μl isopropanol and precipitated at room temperature for 30 min. After centrifugation at 13,000 rpm for 30min, RNA pellets were washed with 80% ethanol, dissolved in ultra-pure water (#10977, Thermo Fisher Scientific), and the RNA concentration was determined using NanoDrop 2000. Ribosomal RNAs was removed by Biotin-labled rRNA probe. Purified and fragmentated RNA was then treated by Illumina TruSeq Standard Kit: Fragmented RNA was reverse transcribed to first chain cDNA random oligo and reverse transcriptase. Second chain cDNA was generated with DNA polymerase I and RNase H. dTTP was replaced by dUTP when Second chain cDNA was generated to increase the chain-specificity. Double chain cDNA was then poly A tailed and ligated with adaptor. After PCR application , samples were sequenced in illumine Hi-seq platform
GEO Accession
GSM4518463