 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Nov 21, 2024 |
| Title |
LMNSC01 human cell line, replicate 2,scRNAseq |
| Sample type |
SRA |
| |
|
| Source name |
brain
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: brain
cell line: LMNSC01
cell type: neural stem cells
genotype: wild type
treatment: no treatment
|
| Treatment protocol |
no treatment
|
| Growth protocol |
LMNSC01 and LMNSC02 cells were grown in T-75 flasks in NeuroCultTM NS-A Basal Medium (Human) (Stem Cell Technologies, Cat# 05750), supplemented either with NeuroCultTM NS-A Proliferation Supplement (Human) (Stem Cell Technologies, Cat# 05751) or NeuroCultTM NS-A Differentiation Supplement (Human) (Stem Cell Technologies, Cat# 05752), basic Fibroblast Growth Factor (bFGF) at concentration 10ng/mL (Stem Cell Technologies, Cat# 78003), Epidermal Growth Factor (EGF) at concentration 10 ng/mL (Stem Cell Technologies, Cat# 78006), and heparin at 0.2x104% concentration (Stem Cell Technologies, Cat# 07980#) under physiological hypoxia conditions at at 4% O2 and 37C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
LMNSC01 and LMNSC02 cells were dissociated from T75 cell culture flasks and counted, and a targeted recovery of 15,000 cells was calculated for each sample by loading a concentration of approximately 15,000 cells.
The library constructed following the manufacturer’s protocol. Library quality control was based on Agilent Tapestation 4200 HS D1000 screentapes (Agilent Technologies, Waldbronn, Germany). Multiplexed library pool was based on Agilent Tapestation 4200 HS D1000 and Kapa Library Quantification Kit for Illumina platforms (Kapa BioSystems, Boston, MA)
Cells were immediately processed with the 10× Genomics Chromium Next GEM Single Cell 3’ v3.1 (Dual Index) kit (10× Genomics, Pleasanton, CA). Samples were loaded, cDNA amplified, and library constructed following the manufacturer’s protocol. Library quality control was based on Agilent Tapestation 4200 HS D1000 screentapes (Agilent Technologies, Waldbronn, Germany). Multiplexed library pool was based on Agilent Tapestation 4200 HS D1000 and Kapa Library Quantification Kit for Illumina platforms (Kapa BioSystems, Boston, MA) and sequenced at shallow depths on Illumina’s iSeq 100 v2 flowcell for 28×10×10×90 cycles for estimated reads per cell. After demultiplexing, libraries were normalized based on their reads per cell. Rebalanced pool QC was based on Agilent Tapestation 4200 HS D1000 and Kapa Library Quantification Kit for Illumina platforms and high depth sequenced on Illumina’s NovaSeq 6000 S4 v1.5 flow cell for 101×11×11×101 cycles.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
10x Genomics
|
| Data processing |
The demultiplexing, libraries were normalized based on their reads per cell. Rebalanced pool QC was based on Agilent Tapestation 4200 HS D1000 and Kapa Library Quantification Kit for Illumina platforms and high depth sequenced on Illumina’s NovaSeq 6000 S4 v1.5 flow cell for 101×11×11×101 cycles.
scRNAseq data was analyzed by Illumina Partek™ Flow™ v12.1.0 bioinformatics software (https://www.partek.com/partek-flow/). Using two replicates for each of LMNSC01 and LMNSC02 cell lines, data were imported into Partek Flow, data were processed based on Illumina Single Cell 3' v3 protocol with estimated 13,705 cells, followed by pre-alignment QA/QC to assess the overall quality of the cells then further filtered by three quality control filters: Mitochondrial reads (>5%), the count of read between 500 - 10,000. Features where the value was <=1.0 in at least 99.9% of the cells, were excluded. PCA was made with 100 principal components, and the variance was set as the features contribute. For the UMAP the local neighborhood size was set at 30, a minimal distance of 0.3, distance metric was set as Cosine, 0 iterations, random generator seed 42, the initialize output values Random, and the number of principal components was set as 20. Normalization with the setting of log2(cpm+1) followed by differential gene expression (DGE) analysis using ANOVA to generate the statistical significant differential genes filtered by |FC|>2 and FDR < 0.001 by comparing between LMNSC01 and LNDSC02 cell lines for downstream analysis of Hierarchical clustering heatmap, gene set enrichment and Pathway analysis. Additionally, an Qiagen Ingenuity Pathway Analysis was done to explore the interactions between the DEGs based on enrichment score calculated relative to a set of background genes relevant for this experiment.
Assembly: GRCh38
Supplementary files format and content: tab-delimited csv file include raw count matrix file
|
| |
|
| Submission date |
Nov 14, 2024 |
| Last update date |
Nov 21, 2024 |
| Contact name |
Yate-Ching Yuan |
| E-mail(s) |
[email protected]
|
| Organization name |
City of Hope
|
| Street address |
1500 East Duarte
|
| City |
Duarte |
| State/province |
CA |
| ZIP/Postal code |
91010 |
| Country |
USA |
| |
|
| Platform ID |
GPL24676 |
| Series (1) |
| GSE281914 |
Single-cell analysis of L-MYC expressing neural stem cells and their extracellular vesicles revealed distinct progeneitor populations with nerogenic potential [scRNA-seq] |
|
| Relations |
| BioSample |
SAMN44747149 |
| SRA |
SRX26727708 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
|
|
|
|
 |
