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Sample GSM2420830

Query DataSets for GSM2420830

Status

Public on Jun 12, 2017

Title

DeltaCspCE_16h_dualRNAseq2

Sample type

SRA

Source name

HeLa cell culture

Organism

Salmonella enterica subsp. enterica serovar Typhimurium str. SL1344

Characteristics

stategy: dual RNA-seq
genotype/variation: deltaCE GFP

Growth protocol

Salmonella was routine grown in LB (220 rpm, 37C), supplemented with appropriate antibiotics. For RIP-seq, cultures were supplemented with 0.2% L-Arabinose at an OD of 0.2. Cultures were grown to OD 2.0 before harvesting RNA. For dual RNA-seq, OD 2 cultures were used to infect HeLa cells at a multiplicity of infection of 5 and centrifuged for 10 minutes. After 30 minutes at 37C, gentamicin was added to the media to kill extracellular bacteria, and RNA was harvested at the listed time points.

Extracted molecule

total RNA

Extraction protocol

For RIP-seq: When the culture reached an OD600 of 2, bacteria were re-suspended in 800 µl of ice-cold lysis buffer (Tris, pH 8.0, 20 mM, KCl 150 mM, MgCl2 1 mM, DTT 1 mM), and disrupted with 1 ml glass beads (BioSpec Products, 0.1 mm diameter) by shaking at 30 Hz for 10 min. The cleared lysate obtained after centrifugation (16000 rcf, 15 min, 4°C) was incubated with 0.5 µl/OD anti-Flag antibody (Sigma, F1804) at 4°C for 30 min followed by incubation with 64 µl pre-washed Protein-A sepharose (Sigma, P6649) for another 30 min. After 5 washes with ice-cold lysis buffer, the sepharose was subjected to Phenol:Chloroform:Isopropanol extraction to isolate RNA (PCI 25:24:1, pH 4.5; Roth, X985.3). For RNA-seq: Total RNA was extracted with hot phenol and chloroform followed by DNase I treatment. For dual RNA-seq: Total RNA from the infected and sorted cells was isolated using the mirVana kit (Ambion).
The samples were rRNA depleted (RNA-seq and dual RNA-seq), then poly(A)-tailed using poly(A) polymerase and the 5’triphosphate (and eukaryotic cap) structures were removed using tobacco acid pyrophosphatase (TAP). Afterwards, an RNA adapter was ligated to the 5’monophosphate of the RNA. First-strand cDNA synthesis was performed using an oligo(dT)-adapter primer and the M-MLV reverse transcriptase (NEB). The resulting cDNA was PCR-amplified to about 20-30 ng/µL using a high fidelity DNA polymerase (barcode sequences for multiplexing were part of the 3’primers). The cDNA library was purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics) and analyzed by capillary electrophoresis (Agilent Technologies).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Description

csp_HeLa_Salmonella_counts.txt

Data processing

Reads were trimmed for a Phred quality of 20 using the FASTX toolkit version 0.0.13
Adapter trimming using Cutadapt version 1.3
Reads were mapped using the READemption pipeline (Förstner et al., 2014), segemehl, and the lack remapper to the SL1344 genome and associated plasmids (NCBI references: NC_016810.1, NC_017718.1, NC_017719.1, NC_017720.1 )
Coverage plots (wig files) and read quantifications were produced by READemption
Genome_build: NCBI references: NC_016810.1, NC_017718.1, NC_017719.1, NC_017720.1
Supplementary_files_format_and_content: text files are tab delimited including raw read counts; RIP-seq and RNA-seq contain fractional read counts for multimapping reads, while dual RNA-seq counts were generated solely from uniquely mapped reads.

Submission date

Dec 09, 2016

Last update date

May 15, 2019

Contact name

Lars Barquist

E-mail(s)

lars.barquist@helmholtz-hiri.de

Organization name

Helmholtz Institute for RNA-based Infection Research

Street address

Josef-Schneider-Straße 2 / Bau D15