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Sample GSM1537644 Query DataSets for GSM1537644
Status Public on Aug 27, 2016
Title Escherichia ProQ Lysate
Sample type SRA
 
Source name bacterial cells
Organism Escherichia coli
Characteristics strain: W3110
rip antibody: Monoclonal ANTI-FLAG^=AE M2 antibody produced in mouse, Sigma, F1804-200UG,SLBG5673V
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.
Growth protocol LB, 37°C, 220 rpm, OD600=0.5, 2.0, 2.0+6 h
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).
 
Library strategy RIP-Seq
Library source transcriptomic
Library selection other
Instrument model Illumina HiSeq 2000
 
Description Total RNA
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
 
Submission date Nov 04, 2014
Last update date May 15, 2019
Contact name Konrad U. Förstner
E-mail(s) foerstner@zbmed.de
Organization name ZB MED - Information Centre for Life Sciences
Department Information Services
Lab Förstner Lab
Street address Gleueler Str. 60
City Cologne
State/province North Rhine-Westphalia
ZIP/Postal code 50931
Country Germany
 
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)
Relations
BioSample SAMN03161928
SRA SRX751174

Supplementary file Size Download File type/resource
GSM1537644_EC-ProQ_Lysate_div_by_3101260.0_multi_by_2692023.0_forward.wig.gz 11.8 Mb (ftp)(http) WIG
GSM1537644_EC-ProQ_Lysate_div_by_3101260.0_multi_by_2692023.0_reverse.wig.gz 12.2 Mb (ftp)(http) WIG
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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