title: GSM8072450: Mtb exposed to nidulalin A containing fungal filtrate (10X MIC) for 6h rep1; Mycobacterium tuberculosis; RNA-Seq
library_construction_protocol: Cells were harvested after 6 h and 12 h exposure and were washed twice with 7H9-ADGNTw broth by centrifuging (4000 rpm, 4°C, 10 min). Cells were resuspended in 0.9 ml TRIzol and were lysed by bead beating using 0.1 mm Zr/Si beads (6000, 2-3 cycles, 30 sec; 5 mins on ice between pulses). Bead beat lysate was centrifuged (13,000 rpm, 1 min, 4°C) to remove the cell debris and 0.3 ml chloroform (Sigma) was added to the supernatant. Tubes were thoroughly shaken, allowed to sit for 5 min and centrifuged (13,000 rpm, 15 min, 4°C) to separate the upper aqueous layer. Total RNA was precipitated using equal volume of 70% ethanol and vortexed for 30 sec. The solution containing RNA was loaded onto spin cartridge columns and the RNA was eluted according to the manufacturer's guidelines (PureLink, Invitrogen) involving on-column DNaseI treatment (Zymo Research). Total RNA was stored in aliquots at -80°C until further assessments. About 100 ng of Total RNA was used to produce mRNA-seq samples using the Illumina TruSeq Stranded Total RNA Gold kit and sequenced as 151 bp paired-end libraries on an Illumina NextSeq 2000 P2.  cDNA prep and mRNA sequencing were done at the sequencing facility of Fredrick National Laboratory for Cancer Research (FNLCR, Fredrick, MD, USA).