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| Status |
Public on Jan 11, 2024 |
| Title |
ALI day 17 - donor 4 |
| Sample type |
SRA |
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| Source name |
human conjunctiva organoids donor 4
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| Organism |
Homo sapiens |
| Characteristics |
cell line: human conjunctiva organoids donor 4
treatment: ALI day 17
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Tissue samples were obtained one month before the processing. At time of sampling, these were immediately minced in the operating room and placed in 1 mL of CellBanker 1 (Amsbio, 11888) on dry ice. The samples were stored at -80 °C until further processing. On the day of the sort, the 4 biopsies were thawed, pooled and washed with 10 mL of AdDMEM+++. To dissociate the samples, cells were resuspended in 1 mL of 0.25% Trypsin/EDTA (Gibco) and 10 U/mL DNase I (Sigma Aldrich, DN25) and incubated in a 37 °C water bath, while checking the advancement of dissociation regularly under the microscope. Using a pre-wet narrowed Pasteur pipette, the cell suspensions were mechanically dissociated by pipetting up and down. The cell suspensions were washed twice with AdDMEM+++. The cell suspensions from 17 day-old ALI cultures were resuspended in 1 mL FACS buffer (PBS-MQ + 10% Fetal bovine serum - Sigma-Aldrich, F7524). The tissue cell suspension was treated for 5 minutes at RT with red blood cell lysis buffer (Sigma Aldrich, 11814389001), before being washed once with AdDMEM+++. Finally, the cell suspension was resuspended in 1 mL FACS buffer. The cell suspensions that were multiplexed (i.e. conditions 1, 2, 3, 4 and 6) were incubated in labelling oligos CMO303, CMO304, CMO305, CMO306 and CMO307, respectively, for 15 minutes at RT. Then, cells were washed with 6 mL FACS buffer and resuspended in 1 mL of FACS buffer. Immediately before the sort, DAPI (ThermoFisher Scientific, D1306) and DRAQ5 (Biostatus, 62251) were added to the cell suspensions. Living cells (that is DAPI- and DRAQ5+) were sorted on a BD Jazz sorter (BD Bioscience) into FACS buffer. 30,000 cells were used for library preparation.
Libraries were prepared using the 10x Genomics Chromium 3’ Gene Expression solution v3.1
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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 |
10x Genomics
LX243
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| Data processing |
Illumina output (bcl files) converted to fastq files using mkfastq (CellRanger v7.0.1)
Alignment, filtering, barcode and UMI counting done using cellranger count (CellRanger v7.0.1)
For tissue samples, SNP-based demultiplexing using souporcell
Assembly: Genome Reference Consortium Human Build 38
Supplementary files format and content: Per sample filtered_feature_bc_matrix output from CellRanger (v7.0.1); includes gene-barcode matrices, genes, barcodes (demultiplexed for LX255_LX256)
Supplementary files format and content: For tissue sample, Souporcell output for k= 4 clusters
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| Submission date |
Sep 05, 2023 |
| Last update date |
Jan 11, 2024 |
| Contact name |
Marie Bannier-Hélaouët |
| E-mail(s) |
m.bannier@hubrecht.eu
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| Organization name |
Hubrecht Institute
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| Street address |
Uppsalalaan 8
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| City |
Utrecht |
| ZIP/Postal code |
3584CT |
| Country |
Netherlands |
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| Platform ID |
GPL24676 |
| Series (1) |
| GSE242382 |
Single-cell characterization of human conjunctiva tissue, organoids and air-liquid interface cultures (10X) |
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| Relations |
| BioSample |
SAMN37285148 |
| SRA |
SRX21637688 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7761923_LX243_barcodes.tsv.gz |
58.7 Kb |
(ftp)(http) |
TSV |
| GSM7761923_LX243_features.tsv.gz |
325.6 Kb |
(ftp)(http) |
TSV |
| GSM7761923_LX243_matrix.mtx.gz |
150.1 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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