title: RNASeq Reads SABiofilmSmall1
design: RNA sequencing was performed by SeqWright (Houston, TX, USA) and the custom strand-specific sequencing libraries, specifically enriched for small RNAs (<200bp), were generated as previously described (40). Briefly, 2 to 5 ?g total RNA was used for preparation of both (whole and small RNA) strand-specific RNA transcriptome sequencing (RNA-seq) libraries. For whole and small RNA libraries, rRNA, including 5S rRNA, was depleted from total RNA using the Ribo-Zero Magnetic kit (Epicentre, Madison, WI). The directional RNA-Seq libraries for whole and small RNA were developed using the NEXTflex directional RNA-Seq (dUTP-based) kit (Bioo Scientific, Austin, TX). For small RNA libraries specifically, ethanol precipitation was used for cleanup steps to promote small RNA retention.  Depleted RNA from small RNA samples were treated with Tobacco Acid Pyrophosphatase (Epicentre, Madison, WI) at 37°C for 60 min to promote correct adapter ligation followed by organic extraction cleanup (with 25:24:1 phenol:chloroform:isoamyl alcohol) and ethanol precipitation of RNA. Small RNA libraries were prepared using the TruSeq Small RNA sample preparation kit for Adapter ligation (Illumina, San Diego, CA) and sequenced using a paired-end protocol and read lengths of 100 nucleotides. Both small and whole RNA-Seq libraries were subjected to the quantification process and pooled for cBot amplification and a subsequent sequencing run with a HiSeq 2000 platform (Illumina, San Diego, CA).