Native cDNA sequencing of E.coli: untreated, replicate 2, 40% rRNA depletion
Ribosomal RNA (rRNA) depletion was performed using the Pan-Prokaryote riboPOOL by siTOOLs, which effectively removes rRNAs from E. coli. For TEX-treated samples, partial digestion of RNAs that are not 5-triphosphorylated (e.g. tRNAs, rRNAs) was achieved by incubation of the RNA with a Terminator 5-Phosphate-Dependent Exonuclease (TEX, Lucigen). Libraries for Nanopore sequencing were prepared from poly(A)-tailed RNAs according to the protocols provided by Oxford Nanopore (Oxford Nanopore Technologies Ltd, Oxford, UK) for direct sequencing of native RNAs (SQK-RNA001, SQK-RNA002), direct cDNA native barcoding (SQK-DCS109 with EXP-NBD104) and PCR-cDNA barcoding (SQK-PCB109) with the following minor modifications: Agencourt AMPure XP magnetic beads (Beckman Coulter) in combination with 1 l of RiboGuard RNase Inhibitor (Lucigen) were used instead of the recommended Agencourt RNAclean XP beads to clean up samples. For reverse transcription, Maxima H Minus Reverse Transcriptase (Thermo Fisher Scientific) was used for all cDNA samples and for the RNA002 samples (SuperScript III Reverse Transcriptase from Thermo Fisher Scientific used for RNA001 sample).