RNA-seq of yersinia enterocolitica delta DksA and ppGpp
RNA-seq transcriptome library was prepared following TruSeqTM RNA sample preparation Kit from Illumina (San Diego, CA) using 2g of total RNA. Shortly, ribosomal RNA (rRNA) depletion instead of poly(A) purification is performed by Ribo-Zero Magnetic kit (epicenter) and then all mRNAs were broken into short (200nt) fragments by adding fragmentation buffer firstly. Secondly double-stranded cDNA was synthesized using a SuperScript double-stranded cDNA synthesis kit (Invitrogen, CA) with random hexamer primers (Illumina). When the second strand cDNA was synthesized, dUTP was incorporated in place of dTTP. Then the synthesized cDNA was 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 size selected for cDNA target fragments of 200bp on 2% Low Range Ultra Agarose followed by PCR amplified using Phusion DNA polymerase (NEB) for 15 PCR cycles. After quantified by TBS380, paired-end RNA-seq sequencing library was sequenced with the Illumina HiSeqTEN (2  150bp read length).The processing of original images to sequences, base-calling, and quality value calculations were performed using the Illumina GA Pipeline (version 1.6), in which 150bp paired-end reads were obtained. A Perl program was written to select clean reads by removing low-quality sequences, reads with more than 14% of N bases (unknown bases) and reads containing adaptor sequences.