 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Oct 30, 2019 |
| Title |
Donor No.5 BD 6h |
| Sample type |
SRA |
| |
|
| Source name |
Pacreatic islets
|
| Organism |
Homo sapiens |
| Characteristics |
agent: Bacteroides dorei (BD)
time point: 6h
|
| Treatment protocol |
Upon arrival, islets were initially incubated for 6-24h at 37°C in CMRL-1066 medium (Gibco, USA) supplemented with 2mM l-glutamine, 1% penicillin-streptomycin, and 10% fetal bovine serum (FBS). After that, individual donor islets were cultured in four-well plates containing FBS-free media at 1000 islet equivalents (IEQs) per well. Each well was exposed to one of the three bacterial strains at 10 bacteria per cell (BpC) for 6h or 24h. Time matched controls were also included using IL-1βb (InvivoGen) at 1ng/ml (positive control) and FBS-free media (negative control).
|
| Growth protocol |
Bacteroides dorei (BD, Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures Cat# 17855), Escherichia coli (EC, Invitrogen Cat# 18258012), and Ruminococcus gnavus (RG, ATCC, Cat# 29149) were purchased from commercial vendors and grown according to standard protocols. Both BD and RG were grown anaerobically in tryptic soy broth supplemented with Oxyrase (Oxyrase Inc.) and resazurin salt (Sigma-Aldrich) while EC was grown aerobically in Luria-Bertani broth. All bacteria were allowed to reach early/mid-stationary phase of growth. At the end of culture, bacteria were fixed in 4% paraformaldehyde (PFA) for 30 minutes, rinsed 3 times with sterile PBS and then reconstituted in sterile physiological saline. To ensure all bacteria were killed, bacterial stocks were used to inoculate additional broth. Absence of bacterial growth 48h after inoculation under standard culturing conditions indicated all bacteria were killed by the PFA exposure. For confirmation of the purity of tested bacteria, genomic sequencing was performed for each bacterial strain
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using Qiagen RNeasy Minikits (Qiagen) according to the manufacturer's recommendations. Next, RNA integrity and concentration were assessed using an Agilent Bioanalyzer 2100 (Agilent) and Nanodrop Spectrophotometer respectively.
cDNA libraries were generated using a SciClone NGS Library Production Robot (Perkin Elmer) and library quality was verified using the Agilent Bioanalyzer.
RNA sequencing was subsequently performed using an Illumina HiSeq2500 system (Illumina, San Diego, CA, USA) to achieve single 100 base pair reads with a read depths of 15-20 million reads per sample.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
Sample_D5_B
|
| Data processing |
Short raw sequence reads were downloaded from the HiSeq2500 server in FASTQ format and individually mapped to the human reference genome (hg38) using TopHat.
The complete bioinformatics analysis pipeline for the RNAseq data was performed using the Tuxedo protocol, which includes TopHat, Cufflinks, Cuffmerge, and Cuffdiff.
Differential gene expression analysis results from the Cufflinks analysis of individual samples were reported as Fragments Per Kilobase of transcript per Million Mapped reads (FPKM) which reports the normalized expression values for a given gene and accounts for multiple reads for an individual fragment.
Transcripts up- and downregulated relative to controls were considered significantly changed only when the false discovery rate (FDR) adjusted p-value (q-value) was less than 0.05.
Relative differences in gene expression were assessed by comparing the log2 fold change values between the islet FPKMs for bacteria-treated and time-matched controls. For easier interpretation of obtained datasets, all log2 fold change values were converted to their equivalent numeric values.
Genome_build: hg38
|
| |
|
| Submission date |
May 16, 2019 |
| Last update date |
Nov 01, 2019 |
| Contact name |
Ahmed M Abdellatif |
| E-mail(s) |
abdellatif_ma@mans.edu.eg
|
| Organization name |
University of Nebraska Medical Center
|
| Department |
Surgery-Transplant
|
| Lab |
Regenerative Medicine
|
| Street address |
42 and Emile
|
| City |
Omaha |
| State/province |
NE |
| ZIP/Postal code |
68198 |
| Country |
USA |
| |
|
| Platform ID |
GPL16791 |
| Series (1) |
| GSE131320 |
Human Islet Response to Selected Type 1 Diabetes Associated Bacteria |
|
| Relations |
| BioSample |
SAMN11785384 |
| SRA |
SRX5867763 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3769115_D5_B_genes_fpkm_annotat.xlsx |
2.8 Mb |
(ftp)(http) |
XLSX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
|
|
|
|
 |
