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| Status |
Public on Jun 29, 2022 |
| Title |
01clH10000tyEMcdCTRLtp22 |
| Sample type |
SRA |
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| Source name |
GABAergic differentiation culture day 22
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| Organism |
Homo sapiens |
| Characteristics |
cell type: hPSC-derived GABAergic inhibitory neurons
disease status: Unaffected
differentiation day: day 22
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| Growth protocol |
hPSCs were dissociated with Accutase (Life Technologies) and plated on Geltrex-coated 12-well plates at 2.1 × 105 cell/cm2 in E6 (STEMCELL Technologies) medium, supplemented with 10µM rock inhibitor Y-27632 (REPROCELL). The next day, Y-27632 was withdrawn, and neural induction was initiated in E6 medium in the presence of 10µM SB421542 (TGF-β receptor kinase inhibitor; Tocris), 100nM LDN193189 (BMP signaling inhibitor; Tocris) and 2µM XAV939 (WNT pathway inhibitor; REPROCELL), referred to as “neural induction medium”. On day 10 of differentiation, neural progenitors were passaged as aggregates by dispase with a 1:3 ratio, and were replated on poly-d-lysine- and laminin-coated plates in a progenitor expansion medium supplemented with Y-27632. The progenitor expansion medium is a 1-to-1 ratio of DMEM/F12 and Neurobasal medium, with 1× N2 supplement and 1 × B27 supplement without vitamin A, 0.5mg/ml BSA, 1× Pen-Strep, and 100µM beta-mercaptoethanol (referred to as “progenitor medium”, all from Life Technologies). Progenitor expansion was allowed from day 10-25, during which cells were replated as aggregates weekly. On day 25 of differentiation, cells were dissociated with Accutase, and seeded at 1.3 × 105 cell/cm2 in neuronal maturation medium supplemented with 10µM Y-27632. Maturation medium is composed of Neurobasal Plus, 1× B27 plus, 1× N2 (all from Life Technologies), 100µM cAMP (Sigma), 20ng/ml BDNF (Peprotech), 20ng/ml GDNF (Peprotech), and 200nM ascorbic acid (Sigma).
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Single-cell suspension was prepared following 10X Genomics Single cell protocols: Cell preparation guide (CG00053, Rev C).
Single Cell Labelling with the BD™ Single-Cell Multiplexing Kits was performed following protocol Doc ID: 210970 Rev. 2.0 23-21340-00
Droplet-based single-cell partitioning and single-cell RNA-Seq libraries were generated using the Chromium Single-Cell 3′ Reagent v2 Kit (10X Genomics, Pleasanton, CA) as per the manufacturer’s protocol based on the 10X GemCode proprietary technology
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
Single Cell 5’ Gene Expression
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| Data processing |
scRNA-seq bcl to fastq conversion using CellRanger (v2.1.1; 10x Genomics) cellranger mkfastq
scRNA-seq mapping using CellRanger (v2.1.1; 10x Genomics) cellranger count. The 98-bp-long read 2 containing the cDNA sequence was mapped using STAR against the human GRCh38 reference genome using CellRanger (v2.1.1; 10x Genomics). refdata-cellranger-GRCh38-1.2.0 was used as a gene model for the alignment and was provided as part of the CellRanger pipeline as a compatible transcriptome reference.
scRNA-seq mapping using CellRanger (v2.1.1; 10x Genomics) cellranger count. The 26-bp-long read 1 was processed to extract cell bar codes and Unique molecular identifiers (UMI). UMIs quantification, GemCode, and cell barcodes filtering based on error detection by Hamming distance were performed as described by Zheng et al
Loupe™ Cell Browser (v2.0.0) was used to view the entire dataset and interactively find significant genes, cell types, and substructure within cell clusters.
Assembly: GRCh38
Supplementary files format and content: .cloupe 10x genomics Loupe™ Cell Browser (v2.0.0) files for interactive exploratory tertiary analysis
Supplementary files format and content: .tar archive containing outputs of cellranger_reanalyse secondary analyis step (clustering, diffexp, pca, tsne)
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| Submission date |
Mar 09, 2022 |
| Last update date |
Jun 29, 2022 |
| Contact name |
Alain Domissy |
| Organization name |
The Scripps Research Institute
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| Department |
Immunology
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| Lab |
Computational Immunology Group
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| Street address |
10550 North Torrey Pines Road
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| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92037 |
| Country |
USA |
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| Platform ID |
GPL18573 |
| Series (1) |
| GSE198138 |
Delayed maturation of Fragile X Syndrome GABAergic neurogenesis revealed by functional and single-cell gene expression analysis |
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| Relations |
| BioSample |
SAMN26542179 |
| SRA |
SRX14417485 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5941764_01clH10000tyEMcdCTRLtp22_analysis.tar.gz |
11.7 Mb |
(ftp)(http) |
TAR |
| GSM5941764_01clH10000tyEMcdCTRLtp22_loupe.cloupe.gz |
9.4 Mb |
(ftp)(http) |
CLOUPE |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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