title: GSM4851786: ∆vtlR/lsrB, OD 0.5, rep2; Agrobacterium fabrum str. C58; RNA-Seq
library_construction_protocol: RNA was extracted via hot-phenol method as described in Wilms et al. (2012, DOI: 10.4161/rna.17212). Samples were treated with RNase-free DNase. RNA libraries were prepared by Novogene. 1 µg of RNA was used. Sequencing libraries were generated with NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) according to manufacturer's instructions. Index codes were added to attribute sequences to each sample. Fragmentation was done with divalent cations in NEBNext First Strand Synthesis Reaction Buffer (5x) at elevated temperature. First strand synthesis was carried out with random hexamer primers and M-MuLV reverse transcriptase. Second strand was synthesized with DNA polymerase I and RNase H. Blunt ends were created from overhangs via exonuclease/polymerase activites. DNA 3' ends were adenylated and NEBNext Adaptors with hairpin loop were ligated. Fragments were purified with the AMPure XP system (Backman Cooulter, USA). The cDNA was incubated at 37 °C for 15 min with 3 µl USER Enzyme (NEB, USA) and then at 95 °C for 5 min.The subsequent PCR was done with Phusion High-Fidelity DNA polymerase, Universail PCR primers and Index (X) Primer. The PCR products were purified via AMPure XP system and the quality was assured via Agilent Bioanalyzer 2100. RNA-Seq (Illumina NovaSeq 6000 platform)