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| Status |
Public on Dec 13, 2023 |
| Title |
H9 WT (Cl18) |
| Sample type |
SRA |
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| Source name |
H9
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| Organism |
Homo sapiens |
| Characteristics |
cell line: H9
cell type: Naive HESCs
library strategy: scRNA-seq
genotype: WT
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| Treatment protocol |
Prior to dissociation cells were washed with PBS and afterwards cells were dissociated with Accutase for 5 minutes at 37C. Afterwards cells were resuspended in MEF media and span down at 1000 rpm for 5 minutes. Cell pallet was washed ones with PBS+0.04% BSA and after 5 minute spin cells were resuspended in PBS+0.04% BSA. To get rid of cell clusters, suspension was strained through 40 micron strainer. Prior to 10X run cell number and viability was assessed by Countess automated cell counter (Thermo).
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| Growth protocol |
Cells used for in this experiment were cultured in 5iLAF naïve ESC medium (1:1 mixture of DMEM/F12 and Neurobasal supplemented with N2, B27, penicillin/streptomycin, nonessential amino acids, GlutaMAX, 0.5% KSR, 0.1mM β-mercaptoethanol, 50ug/ml bovine serum albumin, 20ng/ml _x005F_x005F_x005F_x000B_rhLIF, 20ng/ml Activin A, 8ng/ml FGF2, 1uM MEK inhibitor _x005F_x005F_x005F_x000B_PD0325901 0.5uM B-Raf inhibitor SB590885, 1uM GSK3-β inhibitor IM-12, 1uM Src inhibitor WH-4-023, and _x005F_x005F_x005F_x000B_10uM ROCK inhibitor Y-27632). Both H9 WT and XIST deletion hESCs were cultured in 5% CO2, 5% O2 (Hypoxia) at 37C.
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| Extracted molecule |
total RNA |
| Extraction protocol |
scRNA-seq libraries were generated immediately after cell dissociation using 10X Genomics Chromium instrument and Chromium single cell 3’ reagent kit V3. Each individual run was designed to target 10 000 cells per library. Libraries were generated according to manufacturer’s instructions and library fragment size distribution was determined by BioAnalyzer instrument. Afterwards libraries were pulled together and sequenced on Illumina Novaseq 6000 platform.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
Single cell RNA of Naive hESCs (H9) used as control for XIST KO (Clone18)
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| Data processing |
Align: CellRanger (v3.0.2) count function was used against hg38 (GRCh38-3) genome assembly. To account for mouse feeder cells, we also aligned the reads against the mouse mm10 genome assembly.
Process: Seurat (v3.1.5) was used for further processing and filtering based on multiple criteria: 1) Initial filtering for genes detected in at least three cells, and cells where at least 800 detected genes. 2) Filtering for cells classified as human by CellRanger GEM classification, to remove mouse feeder cells. 3) Mitochondrial RNA was quantified per cell. The R function isOutlier (scater package v1.12.2) was used to identify outliers based on the library size (nmads>3), number of genes (nmads>3), and the percentage of mitochondrial genes in each cell (nmads>1)
Normalize and scale: sctransform function (Seurat package (v3.1.5)) using default parameters
Cluster: Principal Component Analysis was done using Seurat (v3.1.5) function RunPCA on the most highly variable genes. The top 20 principal components were used to find the 20 nearest neighbors of each cell (using FindNeighbors function from Seurat package), following by FindClusters function to identify cell clusters. Clusters were visualized by uniform manifold approximation and projection (UMAP) dimensional reduction technique using the Seurat RunUMAP function.
Assembly: hg38
Supplementary files format and content: Matrix table with raw gene counts for every single cell
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| Submission date |
Aug 22, 2023 |
| Last update date |
Dec 13, 2023 |
| Contact name |
Kathrin Plath |
| Organization name |
UCLA
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| Street address |
BOX 951737, 36-133 CHS
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| City |
Los Angeles |
| State/province |
CA |
| ZIP/Postal code |
90095-1737 |
| Country |
USA |
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| Platform ID |
GPL24676 |
| Series (2) |
| GSE241441 |
XIST directly regulates X-linked and autosomal genes in naïve human pluripotent cells [scRNA-seq] |
| GSE241444 |
XIST directly regulates X-linked and autosomal genes in naïve human pluripotent cells |
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| Relations |
| BioSample |
SAMN37116379 |
| SRA |
SRX21457723 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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