Rna ultra directional kit

RNA was extracted and prepared as above from biological quadruplicates of S. pneumoniae D39. RNA was pooled and analyzed on a Bioanalyzer 2100 (Agilent) to confirm a RIN value > 8 according to the manufacturer’s instructions. RNA was then submitted to the Australian Genome Research Facility (AGRF) for sequencing. In brief, the Epicentre Bacterial Ribozero Kit (Illumina) was used to deplete ribosomal RNA content before the generation of barcoded libraries using Ultra Directional RNA kit (New England Biolabs). Prepared libraries were then sequenced using an Illumina HiSeq2500 with Version 3 SBS reagents and 2 × 100 bp single-end chemistry. Reads were aligned to the S. pneumoniae D39 genome (GenBank accession number NC_008533) using BOWTIE2 version 2.2.6^{84 (link)}. Counts for each gene were obtained using SAMtools version 1.2^{85 (link)} and BEDtools version 2.24.0^{86 (link)}, and differential gene expression was determined using R (DESeq Library) version 3.2.2^{87 (link)}. Transcriptomic data have been deposited in the NCBI Gene Expression Omnibus databank under submission identifier GSE141681.

S. pneumoniae strains were grown in C+Y without added sugars to an OD₆₀₀ of 0.15 and then treated with or without 10 μM AI-2 for 30 min. RNA extraction was performed with a Qiagen RNase minikit according the manufacturer’s protocol. Each sample was derived from three independent cultures (biological replicates) and pooled prior to transcriptome sequencing (RNA-seq) analysis. Total RNA was submitted to the Australian Genome Research Facility (Melbourne, Australia) for sequencing. Briefly, the Epicentre Bacterial Ribozero kit (Illumina) was used to reduce the rRNA content, and then the Ultra Directional RNA kit (New England Biolabs) was used to generate bar-coded libraries. Prepared libraries were then sequenced with the Illumina HiSeq 2500. Trimmed RNA-seq fastq files were then mapped to the D39 reference genome with BOWTIE2 version 2.2.3 (43 (link)). Counts for each gene were obtained with the aid of BEDTools (44 (link)), and differential gene expression was examined with DESeq (45 (link)).

RNA isolated from biological triplicates of wild-type PAO1 and ΔznuA strains was pooled and submitted to the Adelaide Microarray Centre (University of Adelaide) for sequencing. Briefly, the Epicentre Bacterial Ribozero Kit (Illumina) was used to reduce the ribosomal RNA content of the total RNA pool, followed by use of the Ultra Directional RNA kit (New England Biolabs) to generate the barcoded libraries. Prepared libraries were then sequenced using the Illumina HiSeq2500 with Version 3 SBS reagents and 2 × 100 bp paired-end chemistry. Reads were aligned to the P. aeruginosa PAO1 genome (GenBank accession number AE004091.2)26 (link) using BOWTIE2 version 2.2.362 (link). Counts for each gene were obtained with the aid of SAMtools (v 0.1.18)63 (link) and BEDtools64 (link) and differential gene expression was examined using DESeq65 (link); the data has been submitted to GEO (accession number GSE60177).