title: RNAseq of LCM3682 TY control
design: After 14 hours incubation in TY medium, 1/10th volume of ice-cold stop buffer (5% phenol in ethanol) was added to each culture and directly centrifuged at 4C, 8,000 rpm for 4 minutes. Total RNA was extracted using the RiboPureTM kit (Ambion) following the manufacturer's recommendations on 6 biological replicates. Two successive runs of ribosomal RNA subtractions were performed using the Microbe ExpressTM kit (Ambion) following the manufacturer's instructions on a pooled mix of the 6 total RNA samples.Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit (Part# 1004898 Rev. D). Briefly, the first step in the workflow involves fragmentation of mRNA into small pieces using divalent cations under high temperature. Then the cleaved RNA fragments were copied into first-strand cDNA using reverse transcriptase and random primers. This was followed by second-strand cDNA synthesis using DNA Polymerase I and RNase H. These cDNA fragments then went through an end-repair process, the addition of a single 'A' base, and then ligation of the adapters. These products were then purified on a gel to select a size range at 200 bp and enriched by PCR to create the final cDNA library. The efficacy of the library construction was checked in a quality control step that involved measuring the adapter-cDNA size and concentration on an Agilent DNA 1000 chip. Sequencing libraries were denatured with sodium hydroxide and diluted to 6 pM in hybridization buffer for loading onto a single lane of an Illumina HiSeq 2000 flowcell V1.5. Cluster formation, primer hybridization and single-read, 36 sequencing cycles were performed on cBot and HiSeq 2000 (Illumina, San Diego, CA), respectively.