GSM2791493: 4_h_MMC_minusTEX; Caulobacter vibrioides NA1000; RNA-Seq
RNA was extracted with the HotPhenol method and digested with DNaseI For the depletion of processed transcripts, equal amounts of RNA were incubated with Terminator 5'-phosphate-dependent exonuclease (TEX) (Epicentre #TER51020) as previously described (Sharma et al., 2010). The transcripts were not fragmented in order to get mainly sequencing reads of the 5'-end of the transcripts. Libraries for Illumina sequencing of cDNA were constructed by vertis Biotechnology AG, Germany (http://www.vertis-biotech.com/), as described previously for eukaryotic microRNAs (Berezikov et al., 2006) but omitting the RNA size-fractionation step prior to cDNA synthesis. Equal amounts of RNA samples were poly(A)-tailed using poly(A) polymerase. Then, the 5'-triphosphates were removed by applying tobacco acid pyrophosphatase (TAP) resulting in 5'-monophosphat. Afterwards, a RNA adapter was ligated to the 5'-phosphate of the RNA. First-strand cDNA was synthesized by an oligo(dT)-adapter primer and the M-MLV reverse transcriptase. In a PCR-based amplification step using a high fidelity DNA polymerase the cDNA concentration was increased to 20-30 ng/µl. A library-specific barcode for multiplex sequencing was part of a 3'-sequencing adapter.