GSM2265841: CuH-O2-sRNA; Paracoccus denitrificans; RNA-Seq
For RNA extraction, 30 mL of mid exponential phase cells (OD600 ≈ 0.4) was added to 12 mL of ice-cold 95% ethanol/5% phenol (pH= 4.3) (v/v) solution, and incubated on ice for 30 min to stabilize RNA and prevent degradation. RNA was isolated, using the Trizol method according to the protocol described in [9]. Trace DNA contamination was removed using Turbo DNA-free DNase (Ambion) and this was confirmed by PCR amplification of RNA samples using MyFi DNA polymerase (Bioline) according to the manufacturer’s instructions. RNA was quantified spectrophotometrically using a Nanodrop 2000 (Thermo Scientific), and integrity of RNA samples was analyzed using an Experion Automated Electrophoresis platform (Bio-Rad) using RNA StdSens chips (Bio-Rad) according to the manufacturer’s instructions. Library preparation and sequencing were performed by Vertis Biotechnology AG, Germany. Briefly, the total RNA samples were split into two, one was enriched for the small RNA fractions <200 nt (s) specifically using the RNeasy MinElute Cleanup Kit (Qiagen). Ribosomal RNA molecules were depleted from both samples using the Ribo-Zero rRNA Removal Kit for bacteria (Epicentre). The rRNA depleted RNA fractions were first treated with Tobacco Acid Pyrophosphatase (TAP, Epicentre). Afterwards, oligonucleotide adapters were ligated to the 5' and 3' ends of the RNA samples. First-strand cDNA synthesis was performed using M-MLV reverse transcriptase and the 3’adapter as primer. The resulting cDNAs were amplified by PCR 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. The cDNA pool was sequenced on an Illumina NextSeq 500 system using 75 bp read length.