|
| Status |
Public on Nov 25, 2020 |
| Title |
Enterococcus faecium +TEX [ID-005280] |
| Sample type |
SRA |
| |
|
| Source name |
Liquid culture at OD600 2.0
|
| Organism |
Enterococcus faecium Aus0004 |
| Characteristics |
strain/background: AUS0004
rna treatment: +TEX
|
| Growth protocol |
E. faecalis V583 or E. faecium AUS0004 were grown overnight at 37°C on M17 agar plates (Oxoid or Sigma-Aldrich) supplemented with 0.5% glucose. For liquid cultures, single colonies were inoculated into a 25 mL glass tube containing 8.3 mL M17 medium supplemented with 0.5% glucose, in order to keep the final proportions of one third of media to two thirds of oxygen. Cultures were grown overnight at 37°C without agitation. Overnight cultures were back-diluted 1:100 into fresh M17 supplemented with 0.5% glucose and grown at 37°C without agitation to late logarithmic/early stationary phase (optical density (OD)600 of 2.0).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Frozen bacterial cultures were thawed on ice, centrifuged, and cell pellets were resuspended in a lysis solution of 600 µl of 10 mg/ml lysozyme in Tris-EDTA (TE) buffer (pH 8.0) and 60 µl of 10% (w/v) sodium dodecyl sulphate (SDS). Bacterial cells were lysed by placing the samples for 10-12 minutes at 64°C in a water bath. Total RNA was extracted from the lysates using the hot phenol method (Blomberg 1990).
cDNA library construction and sequencing of dRNA-seq samples was performed by Vertis Biotechnology AG, Germany (http://www.vertis-biotech.com/). First, total RNA samples were examined by capillary electrophoresis on a Shimadzu MultiNA microchip electrophoresis system to check RNA quality. Total RNA samples were then fragmented by ultrasound (4 pulses of 30 sec at 4°C) and treated with T4 Polynucleotide Kinase (NEB). Each sample was then divided into two halves and one half was subjected to Terminator exonuclease treatment (+TEX) while the other half remained untreated (-TEX). For cDNA synthesis, the +TEX and -TEX RNA samples were poly(A)-tailed using poly(A) polymerase. Next, RNA 5’ Polyphosphatase (Epicentre) was used to remove the 5’PPP structures. Subsequently, 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 around 10-20 ng/µl using a high fidelity DNA polymerase. PCR cycles (12-15 rounds) were performed, with barcode sequences in the 5’ sequencing adapter. The primers for PCR amplification were designed for TruSeq sequencing according to the instructions of Illumina. The cDNAs were purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics) and were analyzed by capillary electrophoresis. cDNA samples were pooled for sequencing. The library pool was fractionated in the range of 200-600 bp via a differential clean-up with the Agencourt AMPure kit, and an aliquot of the size-fractionated cDNA pool was analyzed by capillary electrophoresis. The cDNA pool was single end sequenced on an Illumina NextSeq 500 system using 1x75 bp read length.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
dRNA-seq
TEX-treated total RNA.
|
| Data processing |
Illumina reads in FASTQ format were trimmed using Cutadapt version 1.12 with the options –m 1 –q 20 to trim 3’ bases with a Phred quality score of less than 20.
Mapping was performed with the READemption pipeline (Förstner et al., 2014) version 0.4.3 using the subcommands “create”, “align” and “coverage”. The “align” command was run with the options -c -r -q -g.
Coverage plots (wig files) were produced by READemption.
Genome_build: E. faecium AUS0004 assembly ASM25094v1
Supplementary_files_format_and_content: Normalized and non-normalized wig files.
|
| |
|
| Submission date |
May 29, 2018 |
| Last update date |
Nov 25, 2020 |
| Contact name |
Lars Barquist |
| E-mail(s) |
[email protected]
|
| Organization name |
Universität Würzburg
|
| Department |
Institute for Molecular Infection Biology
|
| Lab |
RNA Biology
|
| Street address |
Josef-Schneider-Straße 2 / Bau D15
|
| City |
Würzburg |
| State/province |
Bayern |
| ZIP/Postal code |
97080 |
| Country |
Germany |
| |
|
| Platform ID |
GPL25049 |
| Series (1) |
| GSE115009 |
Transcriptome mapping of Enterococcus faecalis and Enterococcus faecium |
|
| Relations |
| BioSample |
SAMN09274826 |
| SRA |
SRX4135910 |
