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| Status |
Public on Apr 18, 2018 |
| Title |
WT exp mRNA |
| Sample type |
SRA |
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| Source name |
E. coli MG1693
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| Organism |
Escherichia coli |
| Characteristics |
strain: MG1693
growth phase: Exponential
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| Growth protocol |
Strains were grown in LB medium (Difco) supplemented with thymine (50 µg/ml) at 37ºC, unless otherwise stated. Overnight cultures of single fresh grown colonies were diluted to an initial OD600 appr. 0.03. Cultures were collected at exponential phase (OD600 appr. 0.5).
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using TRI Reagent (Sigma Aldrich), enriched by depleting small RNAs with GeneJET Purification Kit (Fermentas) and rRNA with MICROBExpres Bacterial mRNA Enrichment Kit (Ambion) and fragmented in alkaline solution (2 mM EDTA and 100 mM Na2CO3 pH 9.2 for 40 min at 95°C) to fragments with size of 24-35 nts. For RPFS, cells were lysed by freeze-rupturing (Retch Mill) and 100 A260 units of ribosome-bound mRNA fraction were directly used for polysomal analysis or subjected to nucleolytic digestion with 10 units/µl micrococcal nuclease (Fermentas) for 10 min at room temperature in buffer with pH 9.2 (10 mM Tris pH 11 containing 50 mM NH4Cl, 10 mM MgCl2, 0.2% triton X-100, 100 µg/ml chloramphenicol and 20 mM CaCl2) to obtain the monosomal fraction. Separation was obtained by sucrose density gradient (15-50% w/v). Subsequently, 20-35-nt RNA fragments from the monosomal fraction were size selected on a denaturing 15% polyacrylamide gel.
RNA-size selection and generation of the cDNA libraries was performed as described {Ingolia, 2009}
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
total RNA
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| Data processing |
Quality trimming using FASTX-toolkit, version 0.0.13.2, parameters -v -t 20 -l 10
Adapter cutting using cutadapt, version 1.8.3, parameters -e 0.1 -O 1 -m 12
Genome mapping using Bowtie, version 1.1.2, parameters for samples 1-2: -v 2 --best --strata -m 1
Read counting using bedtools, version 2.17.0, parameter: -s, the middle nucleotide of each read was taken
Genome_build: U00096.3
Supplementary_files_format_and_content: Read counts were normalized by the length of the unique CDS per kilobase (RPKM) and the total mapped reads per million (RPM)
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| Submission date |
Jun 22, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Irina Chelysheva |
| E-mail(s) |
irina.chelysheva@ndm.ox.ac.uk
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| Organization name |
University of Oxford
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| Department |
NDM
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| Lab |
Centre for Human Genetics
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| Street address |
Roosevelt Dr
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| City |
Oxford |
| ZIP/Postal code |
OX3 7BN |
| Country |
United Kingdom |
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| Platform ID |
GPL14548 |
| Series (1) |
| GSE100373 |
The RNA-binding protein Hfq is important for ribosome biogenesis and affects translation fidelity |
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| Relations |
| BioSample |
SAMN07269355 |
| SRA |
SRX2947190 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2679694_WT_exp_mRNA.tab.gz |
51.7 Kb |
(ftp)(http) |
TAB |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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