title: Bacteroides thetaiotaomicron RNA-seq
design: RNA was isolated from Bt cells ; WT and dma2 were grown overnight in BHI media before being diluted to the equivalent of OD 0.1 in 10mL and grown anaerobically for 4 h at 37C. Four individual overnight and 10-mL culture biological replicates were prepared. Cultures were normalized, and an amount of culture equivalent to an OD600 of 4.0 was pelleted for 90s at 8,000 rpm. Pellets were resuspended on ice in 1 mL TRIzol (Invitrogen) with 10L of 5mg/mL glycogen. Samples were flash frozen and stored at 80C until extraction. Prior to extraction, samples were thawed on ice, then pelleted, and supernatants were treated with chloroform. RNA was extracted from the aqueous phase using the RNeasy minikit (Qiagen, Inc.), and RNA quality was checked by agarose gel electrophoresis and A260/A280 measurements. RNA was stored at 80C with SUPERase-IN RNase inhibitor (Life Technologies) until library preparation. 400ng of total RNA per sample was used to generate cDNA libraries following the RNAtag-Seq protocol. Libraries were PCR-amplified and sequenced using an Illumina NextSeq500 platform, yielding over 7 million reads per sample. Data analysis was performed using our in-house pipeline, Aerobio.