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| Status |
Public on May 17, 2022 |
| Title |
24 hours hfq 1xg control rep 1-1 |
| Sample type |
SRA |
| |
|
| Source name |
3-D co-culture model infected for 24 hours with the S. Typhimurium hfq mutant grown in the RWV in the control orientation
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| Organisms |
Homo sapiens; Salmonella enterica subsp. enterica serovar Typhimurium |
| Characteristics |
tissue: In vitro 3-D co-culture model comprised of human colonic epithelial cells and macrophages
strain: HT-29 (ATCC HTB-38), U937 (ATCC CRL-1593.2), chi_3339
bacterial genotype: delta_hfq
bacterial growth orientation: control
time post-infection: 24 hours
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| Treatment protocol |
For infection studies, 3-D models were counted and evenly seeded into multiwell plates at 1x10^6 cells/well. Cells were infected at a multiplicity of infection (m.o.i.) of ~15-30 for 30 minutes, 3 hours or 24 hours with either WT or hfq mutant S. Typhimurium grown in the RWV. To eliminate extracellular bacteria, gentamicin was added to the samples at concentrations of 50 µg/mL and 10 µg/mL at the 30 minute and 3 hour time points, respectively.
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| Growth protocol |
For RNA-Seq analysis of the bacteria just prior to infection, bacteria were cultured in Lennox Broth (LB) for 24 hr at 37 C and 25 rotations per minute (r.p.m.) in RWV bioreactors positioned in the LFS and control orientations. For infection studies, the 3-D co-culture model (HT-29 and U937 cells) was grown in the RWV bioreactor for 13-14 days in GTSF-2 medium at 10% CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
For RNA-Seq analysis of bacteria alone just prior to infection, wild type and hfq mutant S. Typhimurium were cultured to stationary phase in RWV bioreactors, fixed in RNAlater at a 2:1 ratio (fixative: sample) and stored at -80 C until processing. Bacteria were pelleted and resuspended in 200 µL RNase-free TE buffer (ThermoFisher Scientific) containing 2 mg/mL lysozyme (Sigma-Aldrich). Samples were incubated for 10 min at room temperature with intermittent vortexing to facilitate lysis. Qiazol was added to each sample (700 µL) and incubated at room temperature for 5 min with intermittent vortexing. Samples were transferred into prepared MaXtract tubes, 0.2 µL chloroform added, and shaken for 15 sec to mix. Following a 3 min incubation at room temperature, samples were centrifuged at 12,000 x g for 15 min at 4 C. The aqueous phase was then transferred to an RNase-free microfuge tube and 1.5 volumes of 100% molecular biology grade ethanol (Sigma-Aldrich) was added to each sample and inverted to mix. Samples were applied to a miRNeasy column (Qiagen) and purified with on-column DNase I treatment. Eluted samples were quantified using a Nanodrop spectrophotometer and RNA integrity confirmed using an Agilent Bioanalyzer. For RNA-seq analysis of the host following infection, At the specified time points, infected and time-matched uninfected 3-D co-cultures were fixed in 1 mL Qiazol (Qiagen), vortexed and stored at -80 C until processing. To extract total RNA, samples were thawed, incubated for 5 min at room temperature with intermittent vortexing, and added to a prepared High Density MaXtract tube (Qiagen). To each sample, 0.2 mL chloroform (Sigma-Aldrich) was added and tubes were shaken for 15 seconds. Following a 3 min. incubation at room temperature, samples were centrifuged at 12,000 x g for 15 min. at 4 C. The aqueous phase was then transferred to an RNase-free microfuge tube and 1.5 volumes of 200-proof molecular biology grade ethanol (Sigma-Aldrich) was added and tubes were inverted to mix. Samples were applied to miRNeasy columns (Qiagen) and purified according to manufacturer’s instructions with a 20 min. on-column DNase I treatment. Eluted samples were quantified using a Nanodrop spectrophotometer and DNase-treated a second time using the TURBO DNA-free kit (ThermoFisher). RNA integrity was confirmed on an Agilent Bioanalyzer and all samples had an RNA Integrity Number (RIN) > 9.
cDNA was prepared from total RNA using the Ovation RNA-Seq System V2 via single primer isothermal amplification (NuGEN, 7102-A01) and automated on the Apollo 324 liquid handler (Wafergen). cDNA was quantified on the Nanodrop (Thermo Fisher Scientific) and then sheared to approximately 300 bp fragments using the Covaris M220 ultrasonicator. Libraries were generated using the Kapa Biosystem library preparation kit (KK8201). Fragments were end-repaired and A-tailed as described in the Kapa protocol. Individual indexes and adapters (Bioo, 520999) were ligated onto each separate sample. Adapter-ligated molecules were cleaned using AMPure beads (Agencourt Bioscience/Beckman Coulter, A63883) and amplified with Kapa HIFI enzyme (KK2502). Each library was then analyzed for fragment size on an Agilent Tapestation and quantified by qPCR (KAPA Library Quantification Kit, KK4835) on Quantstudio 5 (ThermoFisher Scientific) before multiplex pooling and sequencing on a 1x75 flow cell on the NextSeq platform (Illumina) at the Arizona State University Genomics Core facility.
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| |
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
NextSeq 550 |
| |
|
| Description |
3-D co-culture model infected for 24 hours with the S. Typhimurium hfq mutant grown in the RWV in the control orientation
fpkm_TPM_sal_human.txt
hfq_1xg_24_2_R1.fastq.gz.merged.fastq.gz
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| Data processing |
bcl2fastq v2.17.1.14 used for basecalling
STAR v2.5.1b used for alignment
Stringtie-1.3.4c was used to report FPKM values (Fragments Per Kilobase of transcript per Million mapped reads), read counts and TPM (Transcripts Per Million).
Differential expression (DE) analysis was performed with EdgeR package from Bioconductor v3.2 in R 3.2.3.
Genome_build: Human: GRCh38.p7 primary assembly; Bacteria: S. Typhimurium LT2 genome
Supplementary_files_format_and_content: tab-delimited text files include FPKM and TPM values
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| |
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| Submission date |
Mar 04, 2020 |
| Last update date |
May 17, 2022 |
| Contact name |
Jennifer Barrila |
| E-mail(s) |
Jennifer.Barrila@asu.edu
|
| Phone |
480-727-9282
|
| Organization name |
Arizona State University
|
| Department |
Biodesign Center for Fundamental and Applied Microbiomics
|
| Street address |
1001 S McAllister Ave
|
| City |
Tempe |
| State/province |
AZ |
| ZIP/Postal code |
85287 |
| Country |
USA |
| |
|
| Platform ID |
GPL28223 |
| Series (1) |
| GSE146347 |
Spaceflight analogue culture enhances the host-pathogen interaction between Salmonella and a 3-D biomimetic intestinal co-culture model |
|
| Relations |
| BioSample |
SAMN14290377 |
| SRA |
SRX7847440 |
