GSM9110473: Exp1 H135N, biol rep 2; Vibrio cholerae O395; RNA-Seq
RNA was isolated using Trizol RNA extraction, then treated with Dnase to remove contaminating genomic DNA. RNA concentration and purity was verified by A260/A280 absorbance readings using a NanoDrop spectrophotometer. Approximately 500ng Total RNA was used for stranded mRNA-seq library preparation by following the NEB Directional RNA library preparation guide with NEBNext rRNA depletion kit v2 (human/mouse/rat) (New England Biolabs, Ipswich, MA). The first step in the workflow involved the depletion of rRNA by hybridization of complementary DNA oligonucleotides, followed by treatment with RNase H and DNase to remove rRNA duplexed to DNA and original DNA oligonucleotides, respectively. Following rRNA removal, the left RNA was fragmented into small pieces using divalent cautions under elevated temperature and magnesium. The cleaved RNA fragments were copied into first strand cDNA using reverse transcriptase and random primers. This was followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. Strand specificity was achieved by replacing dTTP with dUTP in the Second Strand Marking Mix (SMM). The incorporation of dUTP in second strand synthesis effectively quenches the second strand during amplification, since the polymerase used in the assay will not incorporate past this nucleotide. These cDNA fragments then went through an end repair process, the addition of a single 'A' base, and then ligation of the adapters. The products were then purified and enriched with PCR to create the final RNA-Seq library. After RNA-seq libraries were subjected to quantification process, pooled for subsequent 100bp paired end sequencing run with Illumina NovaSeq platform S4 flow cell. After the sequencing run, demultiplexing was employed to generate the fastq file, with the average of ~8-10M reads per sample.