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| Status |
Public on Jun 22, 2021 |
| Title |
POLYseq_Pool |
| Sample type |
SRA |
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| Source name |
Standard cell lines
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| Organisms |
Homo sapiens; Mus musculus |
| Characteristics |
cell line: Mix (3T3, ESH9, mES, HUVEC, Hep G2, Hep G2 + high glucose, B16-F10, MEG-01)
treatment: Hep G2 treated with high glucose (4.5 g/l, 25 mM) for 3 days
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| Treatment protocol |
HepG2 was subject ot high glucose using DMEM + GlutaMAX + 10% FBS for 3 days.
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| Growth protocol |
For HLOs: iPSCs were seeded into 6-well plates (Corning) in mTeSR supplemented with Y-27632 and Laminin-511. Medium was changed to mTeSR alone the following day. Medium was switched to RPMI-1640 (Life Technologies, USA) containing 100 ng/ml Activin A (R&D Systems, USA) and 50 ng/ml bone morphogenetic protein 4 (BMP4; R&D Systems) on the second day. This constitutes day 1 (D1) of differentiation. Medium was switched to RPMI-1640 + 100 ng/ml Activin A + 0.2 % KnockOut Serum Replacement (KOSR; ThermoFisher Scientific) on D2. Medium was switched to RPMI-1640 + 100 ng/ml Activin A + 2.0 % KOSR on D3. Medium was switched to Advanced DMEM/F-12 + B-27 (Gibco, USA) + N2 (Gibco, USA) + 500 ng/ml fibroblast growth factor 4 (FGF-4; R7D Systems) and 3 µM CHIR99021 (R&D Systems) for D4-6, changed daily. A single cell suspension was isolated on D7 using Accutase. Cells were washed and resuspended in growth factor Matrigel at 50,000 cells / 50 µl. Into 6-well VWR plates were plated 50 µl drops. Medium was switched to Enrichment Medium (EP): Advanced DMEM/F12 (Gibco, USA) + 2 % B-27 (Gibco, USA) + 1 % N2 (Gibco, USA) + 1 % HEPES (1 M, Gibco, USA) + 1 % Pen/Strep (ThermoFisher Scientific) + 1 % L-glutamine (ThermoFisher Scientific) + 3 µM CHIR99021 (R&D Systems) + 5 ng/ml FGF2 (R&D Systems) + 10 ng/ml VEGF (Life Technologies) + 20 ng/ml EGF (R&D Systems) + 0.5 µM A83-01 (Tocris) + 50 µg/ml ascorbic acid (Sigma-Aldrich) for D7-10, changed on D7 and D9. Medium was switched to Advanced DMEM/F12 (Gibco, USA) + 2 % B-27 (Gibco, USA) + 1 % N2 (Gibco, USA) + 1 % HEPES (1 M, Gibco, USA) + 1 % Pen/Strep (ThermoFisher Scientific) + 1 % L-glutamine (ThermoFisher Scientific) + 2 µM Retinoic acid (Sigma, USA) for D11-14, changed on D11 and D13. Medium was switched to hepatocyte culture medium (HCM; Lonza, USA) + 10 ng/ml hepatocyte growth factor (HGF; PeproTech, USA) + OSM and changed every other day. HLOs were used between D21 – D24. For cell line culture: 3T3 and B16-F10 were maintained in DMEM + GlutaMAX + 10% FBS, Mouse ES cells were maintaned feeder-free on gelatin in serum-free ES medium + 2i + LIF, ESH9 was maintained feeder-free in mTeSR, HUVECs were maintaned in Loza EGM-2, HepG2 was maintained in MEM + 10% FBS, MEG-01 was maintained in RMPI 1640 + 10% FBS.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Samples were digested using accutase / TrypLE Express into single cell suspensions, resuspended in PBS or DMEM/F-12 + 2 % FBS, and filtered by a 40 µm mesh.
Single cell suspensions were immediately run on the 10x scRNAseq platform following digestion using the Chromium Single Cell 3' v3 (HLOs) or Next GEM 3' v3.1 (POLYseq / Totalseq) kits.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
Mix of standard human and mouse cell lines tagged with POLYseq barcoding library
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| Data processing |
Base call conversion was performed using bcl2fastq v2.20.0
FASTQ files were aligned to either hg19 or mm10 v2 genomes using CellRanger v3.1.0
Read depth normalization, scailing, variable feature identification, PCA analysis, cluster identification, tSNE and UMAP clustering, and hashing demultiplexing was performed using Seurat v3.1 (HLO) / v3.2.2 (POLYseq / TotalSeq). Differential expression was performed using DESeq2 v1.28.0. Pathway analysis was performed by GAGE v2.38.3 on cell clusters normalized by the number of reads and log2 transformed. Pathway visualization was conducted through Pathview v1.28.1 using normalized, log2 transformed data.
Genome_build: hg19 / mm10 v2
Supplementary_files_format_and_content: Matrix table with raw gene counts for every gene / oligo barcode of every cell.
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| Submission date |
Dec 29, 2020 |
| Last update date |
Jun 24, 2021 |
| Contact name |
Andrew Dunn |
| E-mail(s) |
andrew.dunn@cchmc.org
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| Organization name |
Cincinnati Children's Hospital Medical Center
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| Department |
Gastroenterology
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| Lab |
Takanori Takebe
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| Street address |
3333 Burnet Ave R3.543
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| City |
Cincinnati |
| State/province |
Ohio |
| ZIP/Postal code |
45229 |
| Country |
USA |
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| Platform ID |
GPL25526 |
| Series (1) |
| GSE163971 |
POLYseq: A poly(ß-amino ester)-based vector for multifunctional cellular barcoding |
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| Relations |
| BioSample |
SAMN17175477 |
| SRA |
SRX9748941 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4992607_POLYseq_barcodes.tsv.gz |
58.0 Kb |
(ftp)(http) |
TSV |
| GSM4992607_POLYseq_features.tsv.gz |
302.9 Kb |
(ftp)(http) |
TSV |
| GSM4992607_POLYseq_matrix.mtx.gz |
116.3 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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