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| Status |
Public on Jun 19, 2020 |
| Title |
ARID1A KO_undiff_scRNA-seq |
| Sample type |
SRA |
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| Source name |
hESCs H9 cells
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| Organism |
Homo sapiens |
| Characteristics |
cell type source: hESCs H9 cells
passage: 50
status: undifferenciated
genotype/variation: ARID1A KO
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| Treatment protocol |
Cells were dissociated by Corning™ 0.25% Trypsin (Corning™ 25053CI) to single cells.
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| Growth protocol |
For the undifferentiated samples, H9 hESCs were routinely cultured in mTesR medium in a matrigel-coated 6-well plate). For the differentiated samples, H9 hESCs were routinely cultured in mTesR medium (a matrigel-coated 6-well plate). When cells density were 100%, medium were changed to chemically defined medium for cardiac differentiation induction.
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| Extracted molecule |
total RNA |
| Extraction protocol |
H9 hESCs and differentiated hESCs were dissociated by Corning™ 0.25% Trypsin (Corning™ 25053CI) to single cells. Single cell 3’ RNA-seq experiments were conducted using the Chromium single cell system (10x Genomics, Inc) and Illumina sequencers at the Center of Molecular Genetics (CMG) of Indiana University School of Medicine. Cell suspension was first inspected on the Countess II FL (Thermo Fisher Scientific) and under microscope for cell number, cell viability, and cell size. Depending on the quality of the initial cell suspension, the single cell preparation included centrifugation, re-suspension, and filtration to remove cell debris, dead cells and cell aggregates. Single cell capture and library preparation were carried out according to the Chromium Single cell 3’ Reagent kits V2 User Guide (10X Genomics PN-120267, PN-1000009, PN-120262). Appropriate number of cells were loaded on a multiple-channel micro-fluidics chip of the Chromium Single Cell Instrument (10x Genomics) with a targeted cell recovery of 10,000. Single cell gel beads in emulsion containing barcoded oligonucleotides and reverse transcriptase reagents were generated with the v2 single cell reagent kit (10X Genomics). Following cell capture and cell lysis, cDNA was synthesized and amplified. Illumina sequencing libraries were then prepared with the amplified cDNA. The resulting libraries were assessed with an Agilent TapeStation or Bioanalyzer 2100. The final libraries of the undifferentiated samples were sequenced using a custom program on Illumina NextSeq 500/550; and the libraries of the differentiated samples were sequenced on Illumina NovaSeq 6000. 26 bp of cell barcode and UMI sequences, and 91 or 98 bp RNA reads were generated with Illumina NextSeq500/550 or NovaSeq 6000.
Single cell 3’ RNA-seq experiments were conducted using the Chromium single cell system (10x Genomics, Inc) and Illumina sequencers at the Center of Molecular Genetics (CMG) of Indiana University School of Medicine. Cell suspension was first inspected on the Countess II FL (Thermo Fisher Scientific) and under microscope for cell number, cell viability, and cell size. Depending on the quality of the initial cell suspension, the single cell preparation included centrifugation, re-suspension, and filtration to remove cell debris, dead cells and cell aggregates. Single cell capture and library preparation were carried out according to the Chromium Single cell 3’ Reagent kits V2 User Guide (10X Genomics PN-120267, PN-1000009, PN-120262). Appropriate number of cells were loaded on a multiple-channel micro-fluidics chip of the Chromium Single Cell Instrument (10x Genomics) with a targeted cell recovery of 10,000. Single cell gel beads in emulsion containing barcoded oligonucleotides and reverse transcriptase reagents were generated with the v2 single cell reagent kit (10X Genomics). Following cell capture and cell lysis, cDNA was synthesized and amplified. Illumina sequencing libraries were then prepared with the amplified cDNA. The resulting libraries were assessed with an Agilent TapeStation or Bioanalyzer 2100.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
CellRanger 2.1.0 was utilized to process the raw sequence data generated. The filtered gene-cell barcode matrices generated by CellRanger were used for further analysis.
Seurat was used for data normalization, data scaling, graph-based clustering, cluster marker genes idenfificationa.
The CCA function from Seurat was used for integrative analysis of the WT and KO samples.
Genome_build: hg38
Supplementary_files_format_and_content: genes.tsv, matrix.mtx and barcodes.tsv in each tar.gz file
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| Submission date |
Oct 24, 2019 |
| Last update date |
Jun 20, 2020 |
| Contact name |
Lei Yang |
| E-mail(s) |
[email protected]
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| Phone |
(317) 278-5233
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| Organization name |
Indiana University School of Medicine
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| Department |
Department of Pediatrics
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| Street address |
1044 W. Walnut St.
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| City |
Indianapolis |
| State/province |
Indiana |
| ZIP/Postal code |
46202 |
| Country |
USA |
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| Platform ID |
GPL18573 |
| Series (2) |
| GSE139342 |
Single cell RNA-seq revealed different cell types induced by loss of ARID1A in undifferentiated and differentiation (day 10) H9 hESCs. |
| GSE139343 |
Essential and Opposite Roles of ARID1A in Coordinating Human Cardiogenesis and Neurogenesis from Pluripotent Stem Cells |
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| Relations |
| BioSample |
SAMN13108624 |
| SRA |
SRX7051963 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4138490_KO-6.mtx.tsv.tar.gz |
38.1 Mb |
(ftp)(http) |
TAR |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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