|
| Status |
Public on Dec 11, 2015 |
| Title |
24 h mock GFP- replicate 1 |
| 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:mock_GFP_minus:24_h:R1
|
| 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 |
SAMN03470536 |
| SRA |
SRX986578 |
