RNA_Seq of Klebsiella pneumoniae: wild strain C389
First, the rRNA is removed from the total RNA. Then, random fragments of mRNA around 200 bp are generated, and the interrupted mRNA undergoes reverse transcription using dUTP-containing dNTPs and random primers. Next, the End Repair Mix is added to ensure the cDNA ends are flat, followed by the addition of an A base at the 3 end for connecting Y-shaped adaptors. Afterwards, the second strand of cDNA is digested using UNG enzyme. The resulting library is then amplified by PCR for 15 cycles and quantified using TBS380 (Picogreen). The libraries are mixed according to the desired data ratio. Finally, Bridge PCR amplification is performed using cBot to generate clusters. The clusters are then sequenced using the Illumina Hiseq Nova 6000 platform, using 2*150bp/304bp sequencing reads.