GSM8553952: EgtU-Y390A-treated BB10, 21 h, Biol. Rep. 1; Helicobacter pylori PMSS1; RNA-Seq
Total RNA wasisolated using a ZymoDirect-zol™ RNA MiniPrep Plus w/ TRI Reagent®kit. Using the NEBNext rRNA Depletion Kit (Bacteria) (E7850X), bacterial rRNA is depleted starting from 10-1000ng of total RNA by hybridization of rRNA to complementary DNA oligonucleotides, followed by treatment with RNase H and DNase to remove rRNA duplexed to DNA. Next library prep is completed using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (E7760L). Samples are first fragmented at high heat in the presence of divalent cations prior to 1st strand synthesis using random priming. 2nd strand synthesis incorporates dUTPs into the 2nd strand cDNA. End repair creates blunt phosphorylated ends and an adenine overhang is added to the 3 prime end for efficient adapter ligation. The addition of USER enzyme removes the uracil containing strand. The remaining strand is then amplified in a PCR reaction. Indexed libraries that meet appropriate cut-offs for both quantity and quality are quantified by qRT-PCR using a commercially available kit (KAPA Biosystems) and insert size distribution determined with the LabChip GX or Agilent Bioanalyzer. Samples with a yield of ≥0.5 ng/µl are used for sequencing.
GEO
GSM8553952_r1