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Sample GSM1428961 Query DataSets for GSM1428961
Status Public on Dec 04, 2014
Title Hfq-bound RNA from +Vir (virulence-induced) conditions
Sample type SRA
 
Source name bacteria
Organism Agrobacterium tumefaciens
Characteristics strain background: C58
genotype/variation: hfq3xFlag
vir induced with: 0.1 mM acetosyringone
rip antibody: Monoclonal mouse ANTI-FLAG M2 antibody
rip antibody vendor: Sigma
rip antibody cat. #: F1804-200UG
rip antibody batch #: SLBG5673V
Growth protocol Agrobacterium tumefaciens C58 strains were cultivated at 30 °C in YEB complex medium or AB minimal medium (pH 5.5, supplemented with 1% (w/v) glucose) (Schmidt-Eisenlohr et al., 1999). For virulence induction overnight cultures of A. tumefaciens were inoculated in AB medium to an OD600nm of 0.1 and grown for 6 h at 30 °C. Virulence gene expression was induced by addition of 0.1 mM acetosyringone (Sigma-Aldrich, Germany) and further cultivation at 23 °C for 16 h. Non virulence induced cultures were treated with equal volumes of DMSO.
Extracted molecule total RNA
Extraction protocol Co-immunoprecipitation (coIP) experiments of Hfq3xFlag and bound RNAs were based on the procedure described in (Pfeiffer et al., 2007) with minor changes. 100 ml of A. tumefaciens wild-type and hfq3xFlag cultures grown to OD600nm 0.5 and 1.0 in YEB medium or under non-induced (+DMSO) and virulence-induced (+acetosyringone) conditions in AB medium were harvested and resuspended in 2 ml ice-cold lysis buffer (20 mM Tris (pH 7.5), 150 mM KCl, 1 mM MgCl2, 1 mM DTT). Cells were disrupted by French® Press (3 passes, 16,000 psi) and centrifuged at 10,000 x g, 4 °C, for 1 h. 10 ng monoclonal ANTI-3XFLAG® M2 antibody (Sigma-Aldrich, Germany) were coupled to 50 µl Dynabeads® Protein G (ThermoFisher Scientific, Life TechnologiesTM, USA) as described in the instruction manual, and incubated with the supernatant (3 h, 4 °C). Dynabeads® were separated on a magnet and washed 5x with PBS buffer. RNA was isolated using phenol/chloroform/isoamylalcohol and chloroform/isoamylalcohol followed by precipitation with ethanol and sodium acetate. After precipitation remaining DNA was digested by DNaseI (ThermoFisher Scientific, Life TechnologiesTM, USA) and RNA was precipitated as described before.
Preparation of cDNA libraries was performed at Vertis Biotechnology AG (Germany). Equal amounts of RNA samples were poly(A)-tailed and 5’-PPP were removed. The RNA adapter was ligated to the RNA 5’-monophosphate and reverse transcription was performed with oligo(dT)-adapter primers resulting in first-strand cDNA. Higher yields of DNA (20-30 ng µl-1) were gained by further PCR-based amplification using primers designed for TruSeq according to recommendations for Illumina (HiSeq). For multiplex sequencing a library specific barcode was part of the 3’- sequencing adapter. Purification was achieved using the Agencourt AMPure XP kit (Beckman Coulter Genomics, USA), followed by capillary electrophoresis.
 
Library strategy RIP-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 2500
 
Description Hfq-bound RNA
8_Hfq_Flag
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_003062.2,NC_003063.2,NC_003064.2,NC_003065.3
Supplementary_files_format_and_content: wiggle
 
Submission date Jul 07, 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 GPL18897
Series (1)
GSE59123 Identification of Hfq bound RNA by co-immunoprecipitation and RNA-sequencing
Relations
BioSample SAMN02903990
SRA SRX647306

Supplementary file Size Download File type/resource
GSM1428961_8_Hfq_Flag_forward.wig.gz 12.7 Mb (ftp)(http) WIG
GSM1428961_8_Hfq_Flag_reverse.wig.gz 12.9 Mb (ftp)(http) WIG
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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