GSM7900535: RNAseq_60 min replicate iii; Vibrio cholerae; RNA-Seq
RNA was isolated using Tri-reagent. Prepared RNA was digested for DNA contaminations (TurboDNase treatment) and cleaned up using standard nucleic acid precipitation procedures. Depletion of ribosomal RNA was performed by Vertis Biotechnologie AG i.L. company, Freising, Germany. The rRNA depleted RNAs samples were fragmented using ultrasound (4 pulses of 30 sec at 4°C). Then 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. PCR cycles performed and barcode sequences, which are part of the 5' and 3' sequencing adapters, are included in Table 2. The cDNAs were purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics). cDNA from +/- TEX treated RNA: First, the total RNA samples Nr. 31 – 33 were fragmented using ultrasound (4 pulses of 30 sec at 4°C).This was followed by a treatment with T4 Polynucleotide Kinase (NEB). The samples were then split into two halves and one half was subjected to Terminator Exonuclease treatment (+TEX; Lucigen), the other half was left untreated (-TEX). The RNA samples were poly(A)-tailed using poly(A) polymerase. Then, the 5'PPP structures were removed using RNA 5' Polyphosphatase (Lucigen). Afterwards, an RNA adapter was ligated to the 5'-monophosphate of the RNA. First-strand cDNA synthesis was performed using an oligo(dT)-adapter primer and the M-MLV reverse transcriptase. The resulting cDNAs were PCR-amplified to about 10-20 ng/μl using a high fidelity DNA polymerase. PCR cycles performed and barcode sequences, which are part of the 5' and 3' sequencing adapters, are included in Table 2. The cDNAs were purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics).