 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Jun 15, 2024 |
| Title |
R433, scRNAseq |
| Sample type |
SRA |
| |
|
| Source name |
islets
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: islets
Sex: Female
cell type: primary islet cells
bmi: 32.4
diabetesstatus: Non-diabetic
hba1c: NA
age: 44
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Islets were hand picked to minimize acinar tissue contamination, and transferred to a 35 mm dish containing islet culture media. The islets were then re-picked from the 35 mm dish, transferred to a centrifuge compliant tube, and spun down at 150g for 5 min at RT. Supernatant was discarded. To dissociate the islets, islet pellet was resuspended in 2 mL cell dissociation buffer, gently pipetted up and down to mix, transferred to a 35 mm dish, and incubated at 37°C for 2 minutes. Dissociation buffer can be supplemented/replaced with Accutase or Trypsin for more aggressive dissociation if needed. After 2 minutes, islet dissociation was intermittently monitored every 1-2 minutes using a light microscope, with gentle pipetting to aid dissociation. Once dispersion was complete, the cells were strained 2x through a Flomi 40 μm cell strainer to remove any clumps, dispensed into a centrifuge compliant tube, and spun down at 150g for 5 minutes. The cell pellet were resuspended in 1x Ca2+ Mg2+ Free PBS + 0.04% BSA for 10x Chromium Single Cell Applications, and a trypan blue assay was performed to confirm cell viability of >90%, along with cell counting to determine cell concentration of the resuspension.
Library construction was performed according to the manufacturer's instructions (10X Genomics Chromium NEXT GEM Single Cell 3' Gene Expression Kit v3.1). Briefly, cells were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in emulsion (GEM), on a 10X Genomics Chromium Controller. The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contained an Illumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10X Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the appropriately sized fragments were selected with SPRIselect reagent for subsequent library construction. During library construction, Illumina R2 primer sequence, paired-end constructs with P5 and P7 sequences, and a sample index were added.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
10X Genomics
Islets were isolated from pancreases of cadaveric human donors at the Alberta Diabetes Institute IsletCore using standard procedures. The isolated islets were cultured prior to experimental use or distribution in CMRL 1066 (Corning) supplemented with 0.5% BSA (Equitech-Bio), 1% insulin-transferrin-selenium (Corning), 100 U/mL penicillin/streptomycin (Life Technologies), and L-glutamine (Sigma-Aldrich) at 22°C with 5% CO2 at a typical seeding density of 225 IEQ/cm2. Following release from the ADI IsletCore, islets were typically cultured in DMEM (11885, Gibco) supplemented with L-glutamine, 110 mg/l sodium pyruvate, 10% FBS (12483, Gibco), and 100 U/ml penicillin/streptomycin (15140, Gibco) at 37°C with 5% CO2.
|
| Data processing |
Demultiplexing, barcode reading, gene counting, and aggregation were performed by Cell Ranger version 6.1.2
Assembly: GRCh38 (Ensembl release 104)
Supplementary files format and content: Barcodes, features, and matrix files from Cell Ranger
|
| |
|
| Submission date |
Jun 05, 2024 |
| Last update date |
Jun 15, 2024 |
| Contact name |
Patrick E MacDonald |
| E-mail(s) |
pem@ualberta.ca
|
| Organization name |
University of Alberta
|
| Street address |
6-040 Li Ka Shing Centre
|
| City |
Edmonton |
| State/province |
AB |
| ZIP/Postal code |
T6G 2L7 |
| Country |
Canada |
| |
|
| Platform ID |
GPL24676 |
| Series (1) |
| GSE269204 |
Gene expression profile at single cell level of three adult, non-diabetic, human islets |
|
| Relations |
| BioSample |
SAMN41698118 |
| SRA |
SRX24818035 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8308554_R433_1_barcodes.tsv.gz |
31.9 Kb |
(ftp)(http) |
TSV |
| GSM8308554_R433_1_features.tsv.gz |
323.9 Kb |
(ftp)(http) |
TSV |
| GSM8308554_R433_1_matrix.mtx.gz |
51.6 Mb |
(ftp)(http) |
MTX |
| GSM8308554_R433_2_barcodes.tsv.gz |
29.0 Kb |
(ftp)(http) |
TSV |
| GSM8308554_R433_2_features.tsv.gz |
323.9 Kb |
(ftp)(http) |
TSV |
| GSM8308554_R433_2_matrix.mtx.gz |
47.4 Mb |
(ftp)(http) |
MTX |
| GSM8308554_R433_3_barcodes.tsv.gz |
32.1 Kb |
(ftp)(http) |
TSV |
| GSM8308554_R433_3_features.tsv.gz |
323.9 Kb |
(ftp)(http) |
TSV |
| GSM8308554_R433_3_matrix.mtx.gz |
53.5 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
| Raw data are available in SRA |
|
|
|
|
 |
