title: GSM4576391: pWM321 replicate 2; Methanosarcina mazei Go1; RNA-Seq
library_construction_protocol: RNA isolation was performed using TRI reagent following the manufacturer´s protocol followed by DNaseI treatment and phenol-chloroform precipitation cDNA libraries for Illumina sequencing  were constructed by vertis Biotechnologie AG, Germany (http://www.vertis‐biotech.com/) as described previously for eukaryotic microRNA (Berezikov et al., 2006) but omitting the RNA size‐fractionation step prior to cDNA synthesis. In brief, equal amounts of RNA samples were poly(A)‐tailed using poly(A) polymerase. Then, the 5'PPP structures were removed using tobacco acid pyrophosphatase (TAP). Afterwards, an RNA adapter was ligated to the 5 ́‐phosphate of the RNA. First‐strand cDNA was synthesis 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.