alias: GSM1674959
title: GSM1674959: S - starvation conditions; Pectobacterium atrosepticum SCRI1043; ncRNA-Seq
library_construction_protocol: Total RNA was isolated from bacterial cells using the RNeasy Protect Bacteria Mini Kit (Qiagen, USA), according to the manufacturer’s instructions. Contaminating DNA was removed from the samples by DNase (Qiagen) treatment. RNA was quantified using a Qubit fluorometer (Invitrogen, USA). Library construction and strand-specific sequencing were carried out at the Beijing Genomics Institute (BGI-Shenzhen, China; http://www.genomics.cn/en/index), following the manufacturer’s protocols. Briefly, the rRNA was depleted from 1 microgram of total RNA using the Ribo-Zero Magnetic Gold Kit (Epicenter). TruSeq RNA Sample Prep Kit v2 (Illumina) was used for library construction. RNA was fragmented into small pieces using Elute Prime Fragment Mix. First-strand cDNA was synthesized with First Strand Master Mix and Super Script II (Invitrogen) reverse transcription (25°C for 10 min; 42°C for 50 min; 70°C for 15 min). After product purification (Agencourt RNAClean XP Beads, AGENCOURT) the second-strand cDNA library was synthesized using Second Strand Master Mix and dATP, dGTP, dCTP, dUTP mix (1 hour at 16°C). Purified fragmented cDNA was end repaired (30 min at 30°C) and purified with AMPureXP Beads (AGENCOURT). Addition of the poly (A) tail was done with A-tailing Mix (30 min at 37°C) prior to ligating sequencing adapters (10 min at 30°C). The second-strand cDNA was degraded using the Uracil-N-Glycosylase (UNG) enzyme (10 min at 37°C) and the product purified by AMPureXP Beads (AGENCOURT). Several rounds of PCR amplification with PCR Primer Cocktail were performed to enrich the cDNA fragments and the PCR products were purified with AMPureXP Beads (AGENCOURT). Sequencing was performed using the Illumina HiSeqTM 2000 platform with pair-end 90 base reads.
identifiers.namespace: GEO
identifiers.id: GSM1674959