|
|
GEO help: Mouse over screen elements for information. |
|
Status |
Public on Sep 22, 2023 |
Title |
LSC_scRNAseq_3 |
Sample type |
SRA |
|
|
Source name |
Limbal stem cells
|
Organism |
Homo sapiens |
Characteristics |
cell type: Limbal stem cells
|
Growth protocol |
primary KCs were cultured in Keratinocyte Basal Medium supplemented with 100 U/mL Penicillin/Streptomycin, 0.1 mM ethanolamine, 0.1 mM O-phosphoethanolamine, 0.4% bovine pituitary extract, 0.5 μg/mL hydrocortisone, 5 μg/mL insulin and 10 ng/mL epidermal growth factor. Medium was refreshed every other day until the cells were 90% confluent. LSC were cultured in KSFM supplemented with 25 μg/ml Bovine Pituitary Extract, 0.2 ng/ml Epidermal Growth Factor, 0.4 mM CaCl2, 2 mM Glutamine and 100 U/ml Penicillin/Streptomicin. Medium was refreshed every other day untill the cells were 90% confluent.
|
Extracted molecule |
polyA RNA |
Extraction protocol |
A single-cell suspension was made using trypsin. After which cells were filtered using a 40uM filter to remove cell clumps. Cells were stained with 7-AAD. Live cells were selected for and FACs-sorted onto 384-well plates containing primers with unique molecular identifiers, according to the SORT-Seq protocol. Plates were spun down (1200 × g, 1 min, 4 °C) and ERCC spike‐in mix (1:50,000) was dispensed by a Nanodrop into each well. 150 nl of the Reverse Transcription (RT) mix was similarly dispensed into each well. Thermal cycling conditions were set at 4 °C 5 min; 25 °C 10 min; 42 °C 1 h; 70 °C 10 min. Each plate their library was pooled together and the cDNA was purified using AmpureXP beads. Overnight In vitro transcription was carried out at 16 °C, with the lid set at 70 °C. An exonuclease digestion step was performed thereafter for 20 min at 37 °C, followed by fragmentation of the RNA samples. After a beads cleanup, the samples were subjected to library RT and amplification to tag the RNA molecules with specific and unique sample indexes, followed by a final beads cleanup (1:0.8, reaction mix: beads), and the sample cDNA libraries were eluted with DNAse free water. Libraries were quantified using the KAPPA quantification kit following manufacturers protocol after which the plates were sequenced on the Illumina NextSeq 500 for 25 million reads per plate.
|
|
|
Library strategy |
RNA-Seq |
Library source |
transcriptomic single cell |
Library selection |
cDNA |
Instrument model |
Illumina NextSeq 500 |
|
|
Description |
LSC_scRNAseq_3
|
Data processing |
For the matrix files, preprocessing of reads was done automatically with workflow tool seq2science v0.9.2 using the scrna-seq workflow, Single-end reads were trimmed with fastp v0.20.1 with default options. Genome assembly GRCh38.p13 was downloaded with genomepy 0.12.0. The genome and gene annotations was extended with custom regions. Reads were aligned and transformed to bus format with kb-python v0.26.4, a python wrapper for kallisto and bustools. For generating the psuedobulk table, data was prepocessed using the cellseq2 pipeline. Briefly, reads were aligned using star to the GRCh38.p13 genome. After which cells were quality controlled using Seurat, filtering cells on ERCC reads, genes measured and transcripts per cell. After vizualization of the lack of heterogeneity by Umap, pseudo Bulk count data was generated by summing all the cells their UMI counts. Assembly: GRCh38.p13 Supplementary files format and content: tab-delimited text files include gene count values for each plate, for each sample a .mtx matrix file with the reads, a .genes.txt file with the genes corresponding the the matrix file, and a .barcodes.txt file containing the barcodes linked to the matrix file
|
|
|
Submission date |
Jun 24, 2022 |
Last update date |
Sep 22, 2023 |
Contact name |
Jo Huiqing Zhou |
E-mail(s) |
jo.zhou@radboudumc.nl
|
Organization name |
Radboud University
|
Street address |
Geert Grooteplein 26/28
|
City |
Nijmegen |
ZIP/Postal code |
6525GA |
Country |
Netherlands |
|
|
Platform ID |
GPL18573 |
Series (2) |
GSE206923 |
Multi-omics analyses identify transcription factor interplay in corneal epithelial fate determination and disease [scRNA-seq] |
GSE206924 |
Multi-omics analyses identify transcription factor interplay in corneal epithelial fate determination and disease |
|
Relations |
BioSample |
SAMN29336063 |
SRA |
SRX15891801 |
Supplementary file |
Size |
Download |
File type/resource |
GSM6266910_LSC_scRNAseq_2.gzspliced.barcodes.txt.gz |
1.2 Kb |
(ftp)(http) |
TXT |
GSM6266910_LSC_scRNAseq_2.spliced.genes.txt.gz |
108.5 Kb |
(ftp)(http) |
TXT |
GSM6266910_LSC_scRNAseq_2.spliced.mtx.gz |
2.5 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
Processed data are available on Series record |
|
|
|
|
|