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Sample GSM6266910 Query DataSets for GSM6266910
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 SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file
Processed data are available on Series record

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