GSM8920162: isolate vci_091, replicate 1; Mycobacterium tuberculosis; RNA-Seq
Crosslinks were reversed by incubating at 70°C. Cells were lysed in Trizol using bead beating tubes. RNA was extracted by adding chloroform and removing the aqueous upper layer. RNA was precipitated onto carboxylate-modified magnetic beads with isopropanol-GuHCl and beads were washed with ethanol. RNA was resuspended into tris-buffered water. See publication for full details. RNA library preps were prepared for Illumina sequencing using a custom protocol, see publication for complete details. Briefly, cDNA was barcoded using random primers with inline barcodes and an Illumina sequencing adapter. Barcoded, single-stranded cDNA was pooled and a second adapter was added using a splint ligation. Reads corresponding to rRNA were depleted using Taq polymerase's endonuclease activity, combined with a library of guides. Libraries were amplified and sequenced on either a MiSeq or NovaSeq instrument.