title: GSM9102937: UKMD286_5293 deletion mutant biol rep 2; Burkholderia pseudomallei; RNA-Seq
library_construction_protocol: Overnight culture was pelleted and resuspended in TRI Reagent and BCP. Mixtures were vortexed and incubated for 15 min. Aqueous phase was collected and RNA were precipitated using isopropyl alcohol, followed by washing with 75% ethanol. RNA pellet were obtained and resuspended in nuclease free water. The RNA samples were further purified with Pure-NA Biospin Total RNA Extraction Kit (Research Instruments) with on-column DNAseI treatment. A 1μg total RNA was used for following library preparation. The rRNA was depleted from total RNA using rRNA removal Kit. The ribosomal depleted RNA was then fragmented and reverse-transcribed. First strand cDNA was synthesized using ProtoScript II Reverse Transcriptase with random primers and Actinomycin D. The second-strand cDNA was synthesized using Second Strand Synthesis Enzyme Mix (include dACG-TP/dUTP). The purified double-stranded cDNA by beads was then treated with End Prep Enzyme Mix to repair both ends and add a dA-tailing in one reaction, followed by a T-A ligation to add adaptors to both ends.