|
| Status |
Public on Jan 28, 2021 |
| Title |
FibroblastT90Exp2_rep2 |
| Sample type |
SRA |
| |
|
| Source name |
FB_dS_t90
|
| Organism |
Homo sapiens |
| Characteristics |
infection strain: V. parahaemolyticus POR3 ΔvopS
infection time: 90 minutes
cell type: Primary Adult Dermal Fibroblast
|
| Treatment protocol |
V. para cultures were pelleted, resuspended in unsupplemented DMEM and grown at 37°C for 30 minutes to pre-induce T3SS1 expression. Primary Fibroblasts were washed with unsupplemented DMEM and then infected with pre-induced V. para strains at a multiplicity of infection (MOI) of 10. Plates were centrifuged at 1000xg for 5 minutes to synchronize infection and incubated at 37°C, 5% CO2
|
| Growth protocol |
Primary human fibroblasts were seeded onto 6-well plates at a density of 1x10^5 cells/mL and grown for 18-20 hours to ~80% confluency in ATCC Primary Fibroblast Basal Medium plus Low Serum Growth Kit. V. para cultures were grown over night at 30C in MLB and then normalized to an OD600 of 0.2 and subcultured to OD600 = 0.6.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
At 90 minutes post-infection, RNAprotect Cell Reagent (Qiagen) was added. Cells were harvested by scraping, pooled and pellets were resuspended in RLT-plus buffer (Qiagen) and stored at -80C. Cells were lysed with 27G1/2 needles before homogenization with QiAshredder columns (Qiagen). Total RNA was then purified using the RNAeasy Plus Kit (Qiagen).
RNA concentration was determined with a Qubit fluorimeter prior to library prep. 4μg of total DNAse treated RNA were prepared with the TruSeq Stranded Total RNA LT Sample Prep Kit from Illumina. In brief, polyadenylated RNA was purified and fragmented before strand specific cDNA synthesis. cDNA was A-tailed and indexed adapters were ligated. After adapter ligation, samples were PCR amplified and purified with AmpureXP beads, then validated again on the Agilent 2100 Bioanalyzer.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
dS_T90
FB_dS_t90_2
|
| Data processing |
Basecalls performed using bcl2fastq/1.8.4
Reads were trimmed using fastq-mcf (ea-utils/1.1.2-806)
Trimmed reads were mapped using TopHat 2 (2.0.12)
Raw reads were counted using featureCounts (subread/1.4.6)
Normalized counts were generated using edgeR (3.2.4)
Genome_build: hg19
Supplementary_files_format_and_content: tab delimited text file reports normalized counts
|
| |
|
| Submission date |
Sep 20, 2018 |
| Last update date |
Jan 28, 2021 |
| Contact name |
Nicole Janell De Nisco |
| E-mail(s) |
nicole.denisco@utsouthwestern.edu
|
| Organization name |
University of Texas Southwestern Medical Center
|
| Department |
Molecular Biology
|
| Lab |
Orth
|
| Street address |
6000 Harry Hines Blvd, NA5.146, NA5.146
|
| City |
Dallas |
| State/province |
Texas |
| ZIP/Postal code |
75390-9148 |
| Country |
USA |
| |
|
| Platform ID |
GPL16791 |
| Series (1) |
| GSE120273 |
Manipulation of noncanonical Ire1-dependent MAPK signaling by a Vibrio agonist-antagonist effector pair |
|
| Relations |
| BioSample |
SAMN10098272 |
| SRA |
SRX4723282 |
