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| Status |
Public on Sep 11, 2023 |
| Title |
hiPSC-derived islet cells - JUND siRNA |
| Sample type |
SRA |
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| Source name |
iPSC-derived islets
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| Organism |
Homo sapiens |
| Characteristics |
cell type: iPSC-derived islets
developmental stage: S7
treatment: JUND siRNA
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| Treatment protocol |
Stage 7 differentiated beta cells were washed twice with PBS containing 0.5 mM EDTA and incubated in Accumax (SIGMA #A7089) for 8 min at 37 °C after which 50% volume of KnockOut Serum Replacement (ThermoFisher #10828028) was added to stop the reaction. After centrifugation at 400 g for 5 min at room temperature, cells were resuspended in 1 mL HAM’s F-10 medium, supplemented as indicated before (Demine et al., 2020). 75,000 cells in 500 μL medium were seeded per square of a Nunc Lab-Tek II ICC chamber (ThermoFisher). Dispersed human islets and hiPSC-derived islets were transfected with siRNAs for human JUND, (Dharmacon, LQ-003900-00-0002) or human BHLHE41 (Dharmacon, LQ-010043-00-0002). siRNA with no homology to any known mammalian gene was used as negative control (AllStars Negative Control siRNA, Qiagen). siRNA-Lipofectamine RNAiMAX (Invitrogen, Life Technologies) complexes were formed in Opti-MEM and diluted four times in Ham’s F-10 medium without BSA or penicillin-streptomycin. Transfection was done using 30 nM siRNA and Lipofectamine at a final dilution of 1/250. Medium was refreshed after 16 hours and cells were studied after another 48 h.
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| Growth protocol |
Control iPSC lines 1023A (Lytrivi et al. 2021) was differentiated into beta cells using a previously published 7-step protocol (Fantuzzi et al. 2022; De Franco et al. 2020). At the end of the stage 4, cells were seeded into 24-well Aggrewell 400 microwell plates (Stem Cell Technologies) at a density of 0.9 ·10^6 cells per well after which differentiation was carried out as described previously (Cosentino et al. 2018).
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| Extracted molecule |
total RNA |
| Extraction protocol |
hiPSC islets were washed with Versene (Thermo Fisher Scientific, 15040066) and incubated with Accutase (Sigma-Aldrich, A6964-100ML) for 10 min at 37°C. Dissociation was stopped by the addition of PBS supplemented with 1% Bovine Serum Albumin (BSA). Dissociated cells were washed twice in PBS (1%BSA) buffer, centrifuged at 300 rcf for 5 min, filtered using Flowmi 40µm tip strainer (Bel-Art, H13680-0040), counted and adjusted to 1 × 10^6 cells ml–1 cells in PBS (1%BSA) for encapsulation. Cell count and viability were measured using the Luna-Fl automated Fluorescence Cell Counter (Logos Biosystems).
For each siRNA treatment, 6000 cells were loaded onto the 10X Chromium Single Cell Platform (10X Genomics) using the Next GEM Single Cell 3’ library and Gel Bead Kit (v3.1 chemistry) according to manufacturer’s instructions (10x User Guide CG000204, Revision D). RNA quality was assessed using Tapestation (Agilent). Libraries were quantified using Tapestation (Agilent), the Qubit 2.0 (ThermoFisher Scientific) and KAPA Library Quantification Kit for Illumina Platform (KAPA Biosystems) before pooling. Libraries were pooled in equimolar amounts for paired-end sequencing on an Illumina NextSeq 2000 instrument to yield ∼168 million (range 147–195 million) 100-bp-long reads on average per sample.
10X Genomics scRNAseq
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
Raw data was processed using the 10x Genomics CellRanger software (v4.0.0) and mapped to the pre-built human reference genome GRCh38-2020-A. Contamination by background RNA from disrupted cells was estimated and corrected for using SoupX (Young and Behjati 2020) with Seurat identified clusters, and known cell type specific marker genes (GCG, TTR; INS, IAPP; SST; PPY; GHRL; CPA1, CLPS, CPA2, REG1A, CELA3A, CTRB1, CTRB2, PRSS2; KRT19, VTCN1). Cells with less than 200 expressed genes and more than 25% mitochondrial reads were excluded from future analyses. DoubletFinder (v2.0.3)(McGinnis, Murrow, and Gartner 2019) was used with default settings to identify and remove potential doublets. The resulting counts were normalized, scaled and analyzed for principal component analysis (PCA) and uniform manifold approximation and projection (UMAP) on the first 20 principle components (PCs) using 2000 variable genes using Seurat (v3.1.1).
Assembly: GRCh38-2020-A
Supplementary files format and content: Single cell read count matrix (matrix.mtx), Cell barcode (barcodes.tsv), Gene names (features.tsv), raw count matrix (countData.csv), metadata (metadata.csv)
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| Submission date |
Nov 22, 2022 |
| Last update date |
Sep 11, 2023 |
| Contact name |
Vincent Pasque |
| E-mail(s) |
vincent.pasque@kuleuven.be
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| Organization name |
KU Leuven
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| Department |
Development and Regeneration
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| Street address |
Herestraat 49 bus 804
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| City |
Leuven |
| ZIP/Postal code |
3000 |
| Country |
Belgium |
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| Platform ID |
GPL11154 |
| Series (2) |
| GSE218547 |
scRNA-seq of siRNA treated hiPSC-derived islet cells |
| GSE218548 |
Revealing the Key Regulators of Cell Identity in the Human Adult Pancreas |
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| Relations |
| BioSample |
SAMN31837214 |
| SRA |
SRX18348716 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6751705_hiPSC_islets_siJU_barcodes.tsv.gz |
27.9 Kb |
(ftp)(http) |
TSV |
| GSM6751705_hiPSC_islets_siJU_features.tsv.gz |
325.6 Kb |
(ftp)(http) |
TSV |
| GSM6751705_hiPSC_islets_siJU_matrix.mtx.gz |
49.8 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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