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| Status |
Public on Mar 14, 2023 |
| Title |
10X_22_018, no ablation |
| Sample type |
SRA |
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| Source name |
krt4 lineage traced cells in adult zebrafish pancreas
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| Organism |
Danio rerio |
| Characteristics |
genotype: WT
tissue: pancreas
cell type: krt4 lineage traced cells
treatment: no ablation
time: no ablation
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| Treatment protocol |
We selected 6-9 months old fish with 8-10 fish in each condition, We sacrificed the adult fish by putting each one into ice-cold Hanks' Balanced Salt Solution (HBSS, no calcium, no magnesium) for 10 minutes. Next, we used blunt forceps to carefully remove the skin, kidney, eggs (in females), liver and gallbladder to expose the pancreata. This step is critical for avoiding krt4 + cell contamination from other organs. We also removed adipose tissue as much as possible. Then, we cut at the anterior region of the intestinal bulb and hindgut, and transferred the intestine and pancreas together to a new dish. We used blunt dissection tools to carefully separate the pancreata from the intestine. Meanwhile, we also removed the spleen (appearing in dark red), which is also attached to the intestine. We moved and immersed the whole pancreata into 5 mL HBSS kept on ice. For each condition, we pooled 4-8 samples together for an enzymatic digestion with 600 μL 1×TrypLE™ (Thermofisher) supplemented with 60 μL 100× Pluronic™ F68 (Thermofisher) at 37 °C on a shaker at 125 rpm for 45 – 60 minutes to prevent tissue adhesion. We also pipetted the tissue up and down every 5 minutes for better digestion. We added 6 mL precooled 2% BSA to end the digestion and centrifuged the sample at 500 g for 5 minutes. After removing the supernatant, we washed the pellets with 300 μL pre-cooled solution containing 1% BSA, 0.1% Pluronic F-68 and 0.1% DAPI, and used a Corning™ Falcon™ cell strainer (Corning™ 352235) to filter out not fully digested tissues.
During FACS, we selected single-cell populations based on forward scatter and side scatter signals. Next, we performed negative selection with the DAPI channel to remove dead cells and cell debris. Lastly, we used the Cherry channel for the subsequent gating to collect all cells of the krt4-derived lineage in a new tube as the single-cell suspension.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Droplet-based scRNA-seq was performed using the Chromium Single Cell 3′ Library and Gel Bead Kit v3 (10× Genomics) and Chromium Single Cell 3′ Chip G (10× Genomics). Approximately 8,000-10,000 cells from each condition were loaded and encapsulated in a single v3 reaction. GEM generation and library preparation were performed according to manufacturer’s instructions. Cells were partitioned into gel beads in emulsion in the controller for cell lysis and reverse transcription.
The 10× scRNA-Seq libraries were PCR amplified (13 cycles), pooled, denatured, and diluted in prior to paired-end sequencing on a NextSeq 500 according to manufacturer’s recommendations. Sequencing data was aligned to the zebrafish reference genome (GRCz11) with addition of the mCherry sequence using Cell Ranger v5.0.1 (10× Genomics) to generate a gene-by-cell count matrix with default parameters.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
The raw sequencing data was then processed with the ‘count’ command of the Cell Ranger software v5.0.1 (10× Genomics) with the option ‘--expect-cells’ set to 7000 (all other options were used as per default).
Assembly: danRer11
Supplementary files format and content: matrix files
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| Submission date |
Mar 07, 2023 |
| Last update date |
Mar 21, 2023 |
| Contact name |
Olov Andersson |
| E-mail(s) |
olov.andersson@ki.se
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| Phone |
0709644204
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| Organization name |
Karolinska Institute
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| Street address |
Biomedicum Quarter 5D, Tomtebodavagen 16
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| City |
Stockholm |
| State/province |
Other (Non U.S.) |
| ZIP/Postal code |
17165 |
| Country |
Sweden |
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| Platform ID |
GPL20828 |
| Series (1) |
| GSE226841 |
Decoding pancreatic endocrine cell differentiation and beta-cell regeneration in zebrafish |
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| Relations |
| BioSample |
SAMN33843740 |
| SRA |
SRX19740075 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7085292_10X_22_018_filtered_feature_bc_matrix.h5 |
15.9 Mb |
(ftp)(http) |
H5 |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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