GSM6255291: wild-type, RIBO-seq, Replicate 1; Sinorhizobium meliloti 2011; RNA-Seq
RNA was extracted from fractions or cell pellets for total RNA using hot phenol-chloroform-isoamyl alcohol (25:24:1, Roth) or hot phenol (Roth), respectively, as described previously (Sharma et al. 2007). Total RNA was first rRNA-depleted, fragmented then subjected to cDNA library preparation. Size selcted RNA for Riboseq samples were directly subjected to cDNA library preparation. First, an oligonucleotide adapter was ligated to the 3' end of the RNA molecules. First-strand cDNA synthesis was performed using M-MLV reverse transcriptase and the 3' adapter as primer. The first strand cDNA was purified and the 5' Illumina TruSeq sequencing adapter was ligated to the 3' end of the antisense cDNA. The resulting cDNA was PCR-amplified to about 10-20 ng/μl using a high fidelity DNA polymerase. The cDNA was purified  using the Agencourt AMPure XP kit (Beckman Coulter Genomics) and was analyzed by capillary electrophoresis.