title: GSM8655311: ∆lsrB∆oxyR, LB H2O2, biol rep3; Agrobacterium fabrum str. C58; RNA-Seq
library_name: GSM8655311
library_construction_protocol: RNA was extracted using the hot-phenol method in accordance to Wilms et al. (2012, DOI: 10.4161/rna.17212). Residual DNA was removed with RNase-free DNase. RNA libraries were prepared by Novogene using 1 µg of RNA and the NEBNext® Ultra™ RNA Library Prep Kit for Illumina®. Index codes were added to attribute sequences to each sample. The RNA was fragmented with divalent cations in NEBNext First Strand Synthesis Reaction Buffer (5x) at elevated temperature. Then, first strand synthesis was carried out with random hexamer primers and M-MuLV reverse transcriptase. Second strand synthesis was performed with DNA polymerase I and RNase H. Blunt ends were created from overhangs via exonuclease/polymerase activities. DNA 3' ends were adenylated and NEBNext Adaptors with hairpin loop were ligated. Fragments were purified with the AMPure XP system. The cDNA was then incubated at 37 °C for 15 minutes with 3 µl USER Enzyme and then at 95 °C for 5 minutes. The subsequent PCR was performed 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.