Ga iix 75 bp paired end platform

To study changes in gene expression level in Trp and 4FTrp, we have performed strand-specific RNA sequencing on all possible growth conditions of HR23, TR7, together with the parental QB928. Strand-specific paired-end library construction method was described in detail previously (Parkhomchuk et al. 2009 (link)). RNA sequencing was done on the Illumina GA IIx 75 bp paired-end platform (∼200 bp insert size) at the GRC, the University of Hong Kong.
Read counts of transcripts and fragments per kilobase of transcript per million mapped reads were analyzed using Cufflinks 2.1.1(Trapnell et al. 2010 (link); Roberts et al. 2011 (link)), followed by differential gene expression analysis using cuffdiff. Biocyc version 17 (Caspi et al. 2008 (link)), and Pathway Tools 17 (Karp et al. 2010 (link)) were used to look for direct regulates of sigma factors.

DNA was isolated from single-colony overnight cultures of each strain using DNAzol (Chomczynski et al. 1997 (link)). High-throughput DNA sequencing was performed on Illumina GA IIx 75 bp paired-end platform (∼200 bp insert size) at BGI-Shenzhen (BGI) and the Genome Research Center (GRC), University of Hong Kong. Sequence libraries were prepared using Illumina kits with standard protocol. Raw sequencing reads were deposited to National Center for Biotechnology Information (NCBI) Sequence Read Archive (accession ID: SRA057077). The reads from each strain were mapped to the reference QB928 genome (Yu et al. 2012 (link)) using SHRiMP 2.1.1 (David et al. 2011 (link)) (-p opp-in -E -Q –single-best-mapping –half-paired). Gene annotations were originated from B. subtilis str.168 (Barbe et al. 2009 (link)), with refinements described previously (Yu et al. 2012 (link)). Sequencing depths of the different genomes are shown in supplementary table S1, Supplementary Material online.