|
| Status |
Public on Nov 03, 2022 |
| Title |
Grad-seq E. faecium fraction 15 |
| Sample type |
SRA |
| |
|
| Source name |
bacterial culture, gradient fractionation
|
| Organism |
Enterococcus faecium Aus0004 |
| Characteristics |
genotype: wt
growth state: OD 2
|
| Treatment protocol |
DNase I treatment, ERCC spike-in supplementation for Grad-seq. Two polyclonal rabbit antibodies against full-length KhpB were produced by Eurogentec and used for the RIP-seq experiment.
|
| Growth protocol |
E. faecalis V583 and E. faecium AUS004 were grown at 37°C in M17 medium supplemented with 0.5% glucose with one third of media and two thirds oxygen until logarithmic/early stationary phase at an OD600 of 2.0.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Gradient samples were denatured with 1% SDS and RNA was PCI extracted, and ethanol precipitated. 5 µl of the resulting samples were diluted in 45 µl DEPC-treated water. 10 µl were mixedwith 10 µl of a 1:100 diluted ERCC spike-in mix 2 (Thermo Fisher) and subjected to library preparation. In RIP-seq, RNA-protein interaction was denatured by 1% SDS and PCI extraction. RNA was precipitated with ethanol and submitted to library preparation.
The RNA samples were fragmented using ultrasound (4 pulses of 30 s at 4°C) followed by 3′-adapter ligation. First strand cDNA synthesis was performed using M-MLV reverse transcriptase. After purification, the 5′-Illumina TruSeq sequencing adapters were ligated to the 3′end of the antisense cDNA. The resulting cDNA was PCR-amplified to about 10–20 ng/µl by high-fidelity DNA polymerase and purified with Agencourt AMPure XP kit (Beckman Coulter). cDNA samples were pooled with according to input concentration, a size range of 200–550 bp was eluted from a preparative agarose gel. This size-selected cDNA pool was sequenced on an Illumina NextSeq 500 system using 75 ntsingle-end read length.
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
Library strategy: Grad-seq
READemption 0.4.5
Genome_build: NC_004668.1 NC_004669.1 NC_004670.1 NC_004671.1 NC_017022.1 NC_017023.1 NC_017024.1 NC_017032.1
Supplementary_files_format_and_content: wig
|
| |
|
| Submission date |
Feb 10, 2022 |
| Last update date |
Nov 03, 2022 |
| Contact name |
Jörg Vogel |
| E-mail(s) |
[email protected]
|
| Organization name |
Würzburg University
|
| Department |
Institute for Molecular Infection Biology
|
| Lab |
Jörg Vogel lab
|
| Street address |
Josef-Schneider-Straße 2
|
| City |
Würzburg |
| ZIP/Postal code |
97080 |
| Country |
Germany |
| |
|
| Platform ID |
GPL25049 |
| Series (1) |
| GSE196534 |
Enterococcus Grad-seq and KhpB RIP-seq |
|
| Relations |
| BioSample |
SAMN25851550 |
| SRA |
SRX14134069 |
