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| Status |
Public on Aug 27, 2016 |
| Title |
Escherichia WT Lysate |
| Sample type |
SRA |
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| Source name |
bacterial cells
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| Organism |
Escherichia coli |
| Characteristics |
strain: W3110
rip antibody: Monoclonal ANTI-FLAG^=AE M2 antibody produced in mouse, Sigma, F1804-200UG,SLBG5673V
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| Treatment protocol |
50 OD600 of bacteria have been grown to the desired growth stage and harvested by centrifugation. They were lysed in 500 µl of the lysis buffer (20 mM Tris-HCl, pH7.5, 150 mM KCl, 1 mM MgCl2, 1 mM DTT, 1 mM PMSF, 0.2% Triton X100, 20 U/ml DNase I, Thermo Scientific, 200 U/ml SUPERase-IN, Life Technologies). Lysis was carried out on a Retsch MM400 machine at 30 Hz for 10 min in the presence of 750 µl 0.1 mm glass beads. The lysate was cleared by centrifugation at 14,000 g at 4°C for 10 min. The lysate was added 35 µl of monoclonal anti-FLAG M2 antibody (Sigma, #F1804) and rocked for 30 min at 4°C. Then 75 µl of pre-washed Protein A sepharose (Sigma, #P6649) were added and the mixture was rocked for additional 30 min. Afterwards, beads were washed extensively with the lysis buffer, and similar flow-through and wash protein and RNA samples were collected.
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| Growth protocol |
LB, 37°C, 220 rpm, OD600=0.5, 2.0, 2.0+6 h
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| Extracted molecule |
total RNA |
| Extraction protocol |
The equivalent of 5 OD600 was saved for RNA extraction with TriZOL before adding antibodies (lysate RNA sample). For coIP samples, beads were resuspended in the lysis buffer, mixed with an equal volume of phenol:chloroform:isopropanol (25:24:1, pH4.5, Roth) for 20 s and incubated at room temperature for 3 min. After centrifugation, the aqueous phase was precipitated with isopropanol (coIP RNA sample). The purified RNA coIP sample was treated with DNase I (Thermo Scientific) to remove the residual DNA and reisolated with phenol:chloroform:isopropanol. The spike-in RNA (5’P-CUCGUCCGACGUCACCUAGA, IBA) had been added to 40 pg/µl for coIP samples and to 1.6 ng/µl for lysate samples.
RNA-seq libraries were prepared by Vertis AG (Freising-Weihenstephan, Germany). Briefly, RNA was polyadenylated with poly(A) polymerase, 5’-triphosphates were removed with tobacco acid pyrophosphatase followed by ligation of a 5’-adapter. First-strand cDNA synthesis was performed with the use of an oligo(dT) barcoded adapter primer and the M-MVL reverse transcriptase. The resulting cDNA was PCR-amplified with a high fidelity DNA polymerase. cDNA was purified with the Agencourt AMPure XP kit (Beckman Coulter Genomics).
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| Library strategy |
RIP-Seq |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
Total RNA
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| Data processing |
Demultiplexing
Fastq quality trimming using FastX and a cut-off value of 20
Fastq to fasta conversion using FastX
Size filtering: discarding reads shorter than 12 nt (via READemption)
Read mapping using segemehl (via READemption)
Coverage calculation and normalisation (via READemption)
Genome_build: NC_007779.1
Supplementary_files_format_and_content: wiggle
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| Submission date |
Nov 04, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Konrad U. Förstner |
| E-mail(s) |
foerstner@zbmed.de
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| Organization name |
ZB MED - Information Centre for Life Sciences
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| Department |
Information Services
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| Lab |
Förstner Lab
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| Street address |
Gleueler Str. 60
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| City |
Cologne |
| State/province |
North Rhine-Westphalia |
| ZIP/Postal code |
50931 |
| Country |
Germany |
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| Platform ID |
GPL14548 |
| Series (2) |
| GSE62987 |
RIP-seq of RNA associated in vivo with the ProQ, Hfq or CsrA proteins, performed in Salmonella Typhimurium SL1344 or Escherichia coli W3110 |
| GSE62988 |
Partitioning of a RNA interactome by gradient profiling (Grad-seq) |
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| Relations |
| BioSample |
SAMN03161929 |
| SRA |
SRX751176 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1537646_EC-WT_Lysate_div_by_3208904.0_multi_by_2692023.0_forward.wig.gz |
10.3 Mb |
(ftp)(http) |
WIG |
| GSM1537646_EC-WT_Lysate_div_by_3208904.0_multi_by_2692023.0_reverse.wig.gz |
10.8 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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