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| Status |
Public on Oct 21, 2015 |
| Title |
LB RPF |
| Sample type |
SRA |
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| Source name |
Escherichia coli
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| Organism |
Escherichia coli |
| Characteristics |
strain: MC4100
media: LB
sample type: ribosome protected
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| Growth protocol |
E. coli MC4100 strain was cultured at 37°C to mid-log phase (OD600 ~ 0.4) in LB media
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using TRIzol reagent (Invitrogen) and the sample was enriched in mRNA, depleting small RNAs with GeneJET™ RNA Purification Kit (Fermentas) and ribosomal RNA with two cycle of MICROBExpress™Bacterial mRNA Enrichment Kit (Ambion). To probe the RNA structure two µg of enriched mRNA were resuspended in 45 µl of DEPC water and denatured for 3 min at 95°C,refolded at 37 °C, after addition of 10x RNA-structure buffer with pH 7.0 (100 mM Tris, 1 M KCl, 100 mM MgCl2) and digested for 1 min at 37 °C with either 0.05 U RNase V1 (Life Technologies) or a combination of 2 µg RNase A and 5 U RNase T1 (Thermo Scientific). The reaction was stopped by extracting the RNA with phenol-chlorophorm. The RNase A/T1-digested sample was phosphorylated with T4 PNK (NEB) and purified with RNA Clean & Concentrator™ kit (Zymo Research). Both the V1 and A/T1 digested samples were randomly fragmented in buffer with pH 9.2 (100 mM Na2CO3, 2 mM EDTA) for 12 min at 95°C.
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 Genome Analyzer IIx |
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| Description |
ribosome protected
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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 1.2.1, parameters -e 0.1 -O 1 -m 12
Genome mapping using Bowtie, version 0.12.9, parameters for samples 1-5: -v 2 --best --strata -m 1, parameters for samples 6-9: -v 3 --best --strata -m 1
Read using bedtools, version 2.17.0, parameter: -s, for samples 1-5 the middle nucleotide of each read was taken, for samples 6-9 the first nucleotide was taken and the read count was assigned to the nucleotide 5' of the first nucleotide see {Kertesz, 2010} for details
Read counts were normalized by million mapped reads for each nucleotide
Genome_build: strain MG1655, version U00096.2, downloaded from NCBI
Supplementary_files_format_and_content: Column 1 names the nucleotide in the genome, column 2 gives counts for the forward strand, column 3 for the reverse strand, columns are tab separated
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| Submission date |
Dec 03, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Alexander Bartholomaeus |
| E-mail(s) |
bartholomaeus.alexander@gmail.com
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| Organization name |
Helmholtz Centre Potsdam, GFZ German Research Centre for Geosciences
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| Department |
Department 3 Geochemie
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| Lab |
Section 3.7 Geomicrobiology
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| Street address |
Telegrafenberg C
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| City |
Potsdam |
| ZIP/Postal code |
14473 |
| Country |
Germany |
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| Platform ID |
GPL18945 |
| Series (1) |
| GSE63817 |
Secondary structure across the bacterial transcriptome reveals versatile roles in mRNA regulation and function |
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| Relations |
| BioSample |
SAMN03252096 |
| SRA |
SRX795049 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1558079_LB_RPF.tab.gz |
12.5 Mb |
(ftp)(http) |
TAB |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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