title: GSM1406025: MR1 MM TEX(-); Shewanella oneidensis MR-1; RNA-Seq
library_construction_protocol: Bacterial pellets were collected by centrifuging cultures for 10 minutes at 10,000 g at 4 C in RNase-free 50ml polypropylene tubes. Supernatant was immediately poured off and pellets were stored at -80 C. After thawing, RNA was extracted with RNeasy miniprep columns (Qiagen) with the optional on-column DNase treatment. RNA quality was confirmed with an Agilent Bioanalyzer. Equal amounts of total RNA were incubated with terminator 5'-P-dependent exonuclease to degrade transcripts with monophosphate 5' ends (TEX+) or in buffer alone (TEX-). Then we used TAP phosphatase to convert 5'-PPP to 5'-P ends, blocked the 3' ends with sodium periodate and we added a sequencing adaptor onto the 5' end with Ambion T4 RNA ligase. We used random hexamer primers with a sequencing adaptor on their 5' end to obtain first-strand cDNA. We PCR amplified the library to enrich for products that contained both adaptors and to complete the 5' adaptor. A 6-mer index was incorporated into the reverse PCR primer for multiplexing. We purified the PCR products and removed unincorporated nucleotides, primers, and adaptor-only products with AMPure XP Beads (Agencourt).
attributes.tag: GEO Accession
attributes.value: GSM1406025