|
| Status |
Public on Feb 19, 2021 |
| Title |
Ground Infected-1 |
| Sample type |
SRA |
| |
|
| Source name |
In vitro human colonic epithelial cells (HT-29)
|
| Organisms |
Homo sapiens; Salmonella enterica subsp. enterica serovar Typhimurium |
| Characteristics |
strain: HT-29 (ATCC HTB-38), Chi-3339
|
| Treatment protocol |
On Mission Elapsed Day 11, a subset of the HT-29 cultures were infected with ~1x10^7 CFU wild type S. Typhimurium (estimated multiplicity of infection/m.o.i. of ~1-10). The remaining cultures served as time-matched uninfected controls. Six hours post-infection, all cultures were fixed in RNAlater II.
|
| Growth protocol |
HT-29 cells were seeded into collagen-coated hollow fiber bioreactors at Kennedy Space Center at a concentration of 1x10^6 CFU. Cells were cultured using GTSF-2 medium. Wild Type Salmonella Typhimurium strain Chi-3339 (an animal-passaged derivative of wild type SL1344) was prepared from overnight cultures in Dulbecco's Phosphate Buffered Saline (DPBS) stasis buffer at a final concentration of ~1x10^7 CFU/mL. Prepared S. Typhimurium was loaded in to gas permeable FEP bags designated for a subset of the bioreactors. The RNA fixative RNAlater II (40 mL) was separately loaded into FEP bags (one per bioreactor). All bioreactors, bacteria bags and fixative bags were integrated into the Cell Culture Module (CCM) flight hardware, which was then sealed, powered and set to 37 °C and 5% CO2. The CCM was turned over to the KSC integration team at L-18 hours. STL-IMMUNE launched aboard Space Shuttle Discovery (STS-131) on April 5, 2010 at 6:21 am. At approximately L+3 hours, astronauts activated the on orbit programming of the CCM.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted from duplicate samples using the miRNeasy kit (Qiagen). All samples were analyzed in biological duplicate (two separate bioreactors), except for the infected spaceflight sample which was analyzed in technical duplicate from the same bioreactor. Samples were lysed with 700 µl Qiazol (Qiagen), vortexed extensively and passed several times through a 25G needle. After a 5-minute static incubation at room temperature with intermittent vortexing, samples were added to MaXtract High Density tubes (Qiagen) together with 100 µl RNase-free water (Qiagen) and 200 µl chloroform (Sigma-Aldrich). Samples were then processed (including on-column DNase-treatment) according to the manufacturer’s instructions. RNA was quantified using a Nanodrop spectrophotometer and the integrity validated using an Agilent Bioanalyzer 2100 to confirm a RIN of 7 or greater for all samples. One of the uninfected ground control samples (CN122) had a RIN of 6.4.
RNA was fragmented with heat and magnesium to roughly 300 bp using the KAPA HyperPrep RNA-seq kit (Roche). The KAPA Hyper RNA-seq kit and Illumina-compatible adapters (IDT) were used for the remaining library construction. Adapter ligated molecules were cleaned using Kapa pure beads (Kapa Biosciences), amplified with Kapa HIFI enzyme. Each library was then analyzed for fragment size on an Agilent’s Tapestation and quantified by qPCR (KAPA Library Quantification Kit) on Quantstudio 5 (Thermo Fisher Scientific). Libraries were then multiplexed and sequenced on 2x150 flow cell on the NovaSeq platform (Illumina) at University of Colorado Anschutz Medical Campus Genomics and Microarray Core.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
Groud control-cultured HT-29 cells infected for 6 hr with WT S. Typhimurium; Space Shuttle mission STS-131
CN143_S21_L003_R1_001
|
| Data processing |
bcl2fastq v2.17.1.14 used for basecalling
STAR v2.5.1b used for alignment
Cufflinks v2.2.1 was used to report FPKM values (Fragments Per Kilobase of transcript per Million mapped reads) and read counts. TPM (Transcripts Per Million) was calculated by an in-house R script.
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
|
| |
|
| Submission date |
Aug 11, 2020 |
| Last update date |
Feb 19, 2021 |
| 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 |
GPL29006 |
| Series (1) |
| GSE156066 |
Evaluating the effect of spaceflight on the host-pathogen interaction between human intestinal epithelial cells and Salmonella Typhimurium |
|
| Relations |
| BioSample |
SAMN15793450 |
| SRA |
SRX8926834 |
