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Sample GSM1655374 Query DataSets for GSM1655374
Status Public on Dec 11, 2015
Title 24 h WT GFP- replicate 3
Sample type SRA
 
Source name cell culture infection model
Organisms Homo sapiens; Salmonella enterica subsp. enterica serovar Typhimurium str. SL1344
Characteristics human cell line: HeLa-S3 (ATCC CCL-2.2)
infection agent: Salmonella typhimurium SL1344
organism: mixed sample (human cell line HeLa-S3 + Salmonella typhimurium SL1344)
Treatment protocol Infection samples: In vitro infection was carried out following the protocol of (Schulte et al., 2011) with the indicated Salmonella Typhimurium (SL1344) strain in 6-well format. At the indicated time point post-infection, cells were fixed with RNAlater (Qiagen) and sorted for invaded (GFP+) and non-infected (GFP-) host cells using a FACSAria III device (BD). Total RNA from the sorted cells was extracted following the miRvana protocol (Ambion). "0 h" refers to RNA extracted from mixed lysates of extracellular Salmonella and host cells.
Growth protocol HeLa-S3 cells were cultured according the guidelines provided by the ENCODE consortium (http://genome.ucsc.edu/encode/protocols/cell/human/Stam_15_protocols.pdf). Cells were grown in DMEM (Gibco) supplemented with 10% fetal calf serum (FCS; Biochrom), 2 mM L-glutamine (Gibco) and 1 mM sodium pyruvate (Gibco) in T-75 flasks (Corning) in a 5% CO2, humidified atmosphere at 37°C. All further cell lines used in this study (THP-1, CaCo-2, AGS, HEK, MEF and RAW264.7) were cultured in RPMI (Gibco) supplemented with 10% FCS, 2 mM L-glutamine, 1 mM sodium pyruvate in a 5% CO2, humidified atmosphere at 37°C.For the differentiation of bone marrow derived macrophages the marrow of femur and tibia was isolated from 8-12 week old female C57BL/6 wild-type mice and stored in RPMI supplemented with 10% FCS. The cell suspension was centrifuged for 5 min at 250 g and the leukocyte pellet was resuspended in differentiation medium consisting of X-vivo-15 medium (Lonza) supplemented with 10% FCS and 10% L929 conditioned DMEM medium (same composition as above). Cells were cultured for 3 days at 3*106 cells per 10 mL in a T-75 flask. At day 3 another 3 mL of differentiation medium were added and cells were further cultured until day 5. Successful macrophage differentiation was validated by microscopy on day 5 upon which the cells were disattached using a rubber scraper (Sarstedt) and seeded into 6-well plates at 105 cells per well in fresh differentiation medium. Infection was carried out on day 7 as described below.
Extracted molecule total RNA
Extraction protocol miRvana for total RNA (Ambion)
miRvana kit for total RNA (Ambion) or TRIzol (Invitrogen) as indicated
cDNA libraries for Illumina sequencing were generated by Vertis Biotechnology AG, Freising-Weihenstephan, Germany (http://www.vertisbiotech.com/). A minimal amount of ~100 ng of total RNA was required for cDNA library preparation. DNase I-treated total RNA samples were first sheared via ultra-sound sonication (4 pulses of 30 s at 4°C each) to generate on average ~200-400 bp fragmentation products. Then, fragments <20 nt were removed using the Agencourt RNAClean XP kit (Beckman Coulter Genomics). As an internal quality control for this pilot experiment, spike-in RNA (sequence of spike: 5’-AAAUCCGUUCGUACGGGCCC-3’; was 5’-monophosphorylated and gel-purified) was added to a final concentration of 0.5%. The samples were 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 HiSeq 2000
 
Description HeLa-S3:WT_GFP_minus:24_h:R3
Data processing Demultiplexing
FastQ quality trimming using FastX and a cut-off value of 20
Fastq to fasta conversion using FastX (version 0.0.13)
Size filtering: discarding reads shorter than 20 nt (via READemption version 0.3.5)
Read mapping using segemehl version 0.2.0 (via READemption version 0.3.5)
Counting of reads per gene (READemption version 0.3.5)
Genome_build: Human: hg19/GRCh37; Salmonella enterica Typhimurium SL1344: NC_016810.1, NC_017718.1, NC_017719.1, NC_017720.1
Supplementary_files_format_and_content: CSV (tab separated), Number of reads per gene and condition
 
Submission date Apr 10, 2015
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 GPL20050
Series (2)
GSE60144 Dual RNA-seq of diverse human, mouse and pig cell-types infected with various Salmonella strains
GSE67757 Dual RNA-seq – Pilot Dual RNA-seq
Relations
BioSample SAMN03470573
SRA SRX986596

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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