title: The transcriptome of Bacillus velezensis GUIA: cultured without waterborne polyurethane for 4 days
design: (1) Library preparation for strand-specific transcriptome sequencingA total amount of 3 g RNA per sample was used as input material for RNA sample preparation. Sequencing libraries were generated using NEBNext Ultra.  Directional RNA Library Prep Kit for Illumina (NEB, USA) following manufacturers recommendations and index codes were added to attribute sequences to each sample. rRNA is removed using a specialized kit that leaves the mRNA.  Fragmentation was performed with divalent cations under high temperature in NEBNext First Strand Synthesis Reaction Buffer. First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNaseH-). The second strand cDNA was then synthesized using DNA Polymerase I and RNase H. In the reaction buffer, dNTPs with dTTP were replaced by dUTP. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3 ends of DNA fragments, NEBNext Adaptor with hairpin loop structure was ligated to prepare for hybridization. For selecting cDNA fragments of preferentially 150~200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 L USER Enzyme (NEBUSA) was used with size-selected, adaptor-ligated cDNA at 37  C for 15 min followed by 5 min at 95  C before PCR. Then PCR was performed with the High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system.(2) Clustering and sequencingFollowing the manufacturers instructions, the clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia). After cluster generation, the library preparations were sequenced on an Illumina Hiseq platform and paired-end reads were generated.