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| Status |
Public on Oct 03, 2023 |
| Title |
PPC126 |
| Sample type |
SRA |
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| Source name |
iPSC-derived pancreatic progenitor cells
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| Organism |
Homo sapiens |
| Characteristics |
library name: 07493f27-f1b4-4ad6-a3fd-a4419ad496f0
cell type: iPSC-derived pancreatic progenitor cells
time: day15
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| Growth protocol |
The iPSC lines were differentiated into PPCs using the STEMdiffTM Pancreatic Progenitor Kit (StemCell Technologies) protocol with minor modifications. Briefly, iPSC lines were thawed into mTeSR1 medium containing 10 µM Y-27632 ROCK Inhibitor (Selleckchem) and plated onto one well of a 6-well plate coated with Matrigel. iPSCs were grown until they reached 80% confluency 95 and then passaged using 2mg/ml solution of Dispase II (ThermoFisher Scientific) onto three wells of a 6-well plate (ratio 1:3). To expand the iPSC cells for differentiation, iPSCs were passaged a second time onto six wells of a 6-well plate (ratio 1:2). When the iPSCs reached 80% confluency, cells were dissociated into single cells using Accutase (Innovative Cell Technologies Inc.) and resuspended at a concentration of 1.85 x 106 cells/ml in mTeSR medium containing 10 µM Y-27632 ROCK inhibitor. Cells were then plated onto six wells of a 6-well plate and grown for approximately 16 to 20 hours to achieve a uniform monolayer of 90-95% confluence (3.7 x 106 cells/well; about 3.9 x 105 cells/cm2). Differentiation of the iPSC monolayers was initiated by the addition of the STEMdiffTM Stage Endoderm Basal medium supplemented with Supplement MR and Supplement CJ (2 ml/well) (Day 1, D1). The following media changes were performed every 24 hours following initiation of differentiation (2 ml/well). On D2 and D3, the medium was changed to fresh STEMdiffTM Stage Endoderm Basal medium supplemented with Supplement CJ. On D4, the medium was changed to STEMdiffTM Pancreatic Stage 2-4 Basal medium supplemented with Supplement 2A and Supplement 2B. On D5 and D6, the medium was changed to STEMdiffTM Pancreatic Stage 2-4 Basal medium supplemented with Supplement 2B. From D7 to D9, the medium was changed to STEMdiffTM Pancreatic Stage 2-4 Basal medium supplemented with Supplement 3. From D10 to D14, the medium was changed to STEMdiffTM Pancreatic Stage 2-4 Basal medium supplemented with Supplement 4. On D15, cells were dissociated with Accutase and then collected, counted, and processed for data generation. iPSC-PPC cells were cryopreserved in CryoStor® CS10 (StemCell Technologies) until sequencing.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated from total-cell lysates using the Quick-RNATM MiniPrep Kit (Zymo Research) with on-column DNAse treatments. RNA was eluted in 48 µl RNAse-free water and analyzed on a TapeStation (Agilent) to determine sample integrity. All iPSC-PPC samples had RNA integrity number (RIN) values over 9.
Illumina TruSeq Stranded mRNA libraries were prepared according to the manufacturer’s instructions and sequenced on NovaSeq6000 for 101bp paired-end sequencing.
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| Library strategy |
ssRNA-seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
Cells were collected at day 15 of differentiation for sequencing
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| Data processing |
RNA-seq reads were aligned with STAR (2.7.3) 101 to the hg19 reference using GENCODE version 34 hg1996 splice junctions with default alignment parameters and the following adjustments: -outFilterMultimapNmax 20, -outFilterMismatchNmax 999, -alignIntronMin 20, -alignIntronMax 1000000, -alignMatesGapMax.
We obtained common bi-allelic and exonic variants from the 1000 Genomes Phase 3 panel 103 with minor allele frequencies between 45% and 55% and predicted their genotypes in the 107 bulk RNA-seq samples using mpileup and call functions in BCFtools (1.9.0). Then, we used the genome command in plink to estimate the identity-by-state (IBS) between each pair of bulk RNA-seq and WGS samples. VCF containing WGS results for IPSCORE subjects were downloaded from dbGaP phs001325.v3.
TPM and relative isoform usage was estimated for each gene in each RNA-seq sample using RSEM (version 1.2.20) with the following options –seed 3272015 –estimate-rspd –paired-end –forward-prob.
Assembly: GENCODE hg39 version 34
Supplementary files format and content: rsem.genes.results - gene expression quantification
Supplementary files format and content: rsem.isoforms.results - isoform expression quantification
Supplementary files format and content: plink.genome - sample identity results from plink. Each sample is matched to the WGS sample with PI_HAT > 0.95.
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| Submission date |
Jan 14, 2023 |
| Last update date |
Jun 07, 2024 |
| Contact name |
Kelly Ann Frazer |
| E-mail(s) |
kafrazer@health.ucsd.edu
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| Phone |
650 288-9391
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| Organization name |
UC San Diego
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| Department |
Pediatrics
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| Lab |
Frazer
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| Street address |
9500 Gilman Drive
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| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92093 |
| Country |
USA |
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| Platform ID |
GPL24676 |
| Series (1) |
| GSE182758 |
eQTL mapping of fetal-like pancreatic progenitor cells |
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| Relations |
| BioSample |
SAMN32738673 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6934737_07493f27-f1b4-4ad6-a3fd-a4419ad496f0_plink.genome.gz |
14.8 Kb |
(ftp)(http) |
GENOME |
| GSM6934737_07493f27-f1b4-4ad6-a3fd-a4419ad496f0_rsem.genes.results.gz |
1.8 Mb |
(ftp)(http) |
RESULTS |
| GSM6934737_07493f27-f1b4-4ad6-a3fd-a4419ad496f0_rsem.isoforms.results.gz |
3.8 Mb |
(ftp)(http) |
RESULTS |
| Processed data provided as supplementary file |
| Raw data not provided for this record |
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