RNA-Seq of the mutant M246-2: Tn5 transposon mutant derived from the strain GXGL-4A
RNA-Seq library was constructed using the TruSeqTM RNA sample preparation kit from Illumina (San Diego, CA, USA) and 2 g of total RNA. Ribosomal RNA (rRNA) depletion instead of poly(A) purification was carried out by Ribo-ZeroTM Magnetic Kit from Epicentre (Madison, WI, USA), and then mRNAs were broken into short (200 nt) fragments by adding fragmentation buffer firstly. Secondly double-stranded cDNA was synthesized using a SuperScript double-stranded cDNA synthesis kit (Invitrogen, CA, USA) with random hexamer primers (Illumina, CA, USA). When the second strand cDNA was synthesized, dUTP was incorporated in place of dTTP. Subsequently, the synthesized cDNAs were subjected to end-repair, phosphorylation and A base addition according to Illuminas library construction protocol. The second strand cDNA with dUTP was recognized and degraded by UNG enzyme. Libraries were constructed using size-selected cDNA target fragments of 200 bp on 2% agarose and followed by PCR amplification using Phusion DNA polymerase (NEB) for 15 PCR cycles. After quantified by TBS380, the paired-end RNA-Seq libraries were sequenced on the Illumina HiSeq X Ten platform (2  150 bp read length).