RNA-Seq of Weissella koreensis KCKM 0130: 33C, 24h
Total RNA concentration was calculated by Quant-IT RiboGreen (Invitrogen, #R11490). To assess the integrity of the total RNA, samples are run on the TapeStation RNA screentape (Agilent). Only high-quality RNA preparations, with RIN greater than 7.0, were used for RNA library construction.A library was independently prepared with 1ug of total RNA for each sample by Illumina TruSeq Stranded mRNA Sample Prep Kit (Illumina, Inc., San Diego, CA, USA, #20020595). As first step, bacterial rRNA depleted samples were prepared by using the NEBNext rRNA Depletion kit (Bacteria) (NEB). After the rRNA is depleted, the remaining RNA is fragmented into small pieces using divalent cations under elevated temperature. The cleaved RNA fragments are copied into first strand cDNA using SuperScript II reverse transcriptase (Invitrogen) and random primers. This is followed by second strand cDNA synthesis using DNA Polymerase I, RNase H and dUTP. These cDNA fragments then go through an end repair process, the addition of a single A base, and then ligation of the adapters. The products are then purified and enriched with PCR to create the final cDNA library.The libraries were quantified using KAPA Library Quantification kits for Illumina Sequencing platforms according to the qPCR Quantification Protocol Guide (KAPA) and qualified using the TapeStation D1000 ScreenTape (Agilent). Indexed libraries were then submitted to an Illumina NovaSeq (Illumina, Inc., San Diego, CA, USA), and the paired-end (2100 bp) sequencing was performed by the Macrogen Incorporated.