Pokkaliibacter plantistimulans L1E11-WT cultured in Reasoner's 2A broth + 50% sea water Replicate-1
A modified protocol is used to prepare the libraries for total RNA sequencing (figure 1). The first stepfor purifying the poly-A containing mRNA molecules using oligo-dT attached magnetic beads isomitted. Instead, the first step involves the removal of ribosomal RNA of cytoplasmic andmitochondrial origin using biotinylated, target-specific oligos combined with rRNA removal beads.Following purification, the RNA is fragmented using divalent cations under elevated temperature.Next, the cDNA is synthesized using Reverse transcriptase and random hexamers in a first strandsynthesis reaction. Subsequently, the cDNA is converted to double stranded cDNA where Uracil isadded instead of Thymine. The strand specificity is preserved by a USER enzyme based digestion ofsecond strand thereby leaving one functional strand which maps to the DNA strand which it wastranscribed from. The USER digested single strand molecules are enriched and indexed in a limitedcycle PCR followed by AMPure bead purification to create final cDNA library for sequencing.