NCBI Logo
GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
          Go
Sample GSM5888818 Query DataSets for GSM5888818
Status Public on Nov 03, 2022
Title Grad-seq E. faecium fraction 9
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 SAMN25851556
SRA SRX14134063

Supplementary file Size Download File type/resource
GSM5888818_ID-004783-Gr_2_0054_9_div_by_23154917.0_multi_by_4978489.0_forward.wig.gz 6.8 Mb (ftp)(http) WIG
GSM5888818_ID-004783-Gr_2_0054_9_div_by_23154917.0_multi_by_4978489.0_reverse.wig.gz 8.7 Mb (ftp)(http) WIG
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

| NLM | NIH | GEO Help | Disclaimer | Accessibility |
NCBI Home NCBI Search NCBI SiteMap