GSM1200040: G20_LS4D; Desulfovibrio alaskensis G20; RNA-Seq
We used Terminator 5'-Phosphate-Dependent Exonuclease (Epicentre) to try to remove 5'-monophosphate (degraded) transcripts and then converted 5'-triphosphate ends to 5'-monophosphate ends with RNA 5' Polyphosphatase (Epicentre).  Between each enzymatic reaction of this library, RNA was purified using Agencourt RNAClean XP beads. Molecules smaller than 100 nucleotides and unligated adaptors were mostly lost in these clean-up reactions.  We then 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 size-selected products of 150-500 bases from an agarose gel. We PCR amplified the library to enrich for products that contained both adaptors and to complete the 5' adaptor.  We purified the PCR products and removed unincorporated nucleotides, primers, and adaptor-only products with AMPure XP Beads (Agencourt).