 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Jan 15, 2020 |
| Title |
L75P-9_d103-2 |
| Sample type |
SRA |
| |
|
| Source name |
stem cell derived retinal organoid
|
| Organism |
Homo sapiens |
| Characteristics |
days in culture: 103
cell line (all ipsc): L75Pfs clone 9
genotype: L75Pfs
|
| Growth protocol |
Embryoid bodies (EB) were lifted with either 2 mg ml-1 dispase or ReLeSR and weaned into Neural Induction Media (NIM: DMEM:F12 1:1, 1% N2 supplement, 1x MEM nonessential amino acids (MEM NEAA), 1x GlutaMAX and 2 mg ml-1 heparin (Sigma)) over the course of 4 days. On day 6 (d6), 1.5 nM BMP4 (R&D Systems) was added to fresh NIM and on d7, EBs were plated on Matrigel at a density of 200 EBs per well of a 6-well plate. Half the media was replaced with fresh NIM on d9, d12 and d15 to gradually dilute the BMP4 and on d16, the media was changed to Retinal Differentiation Media (RDM: DMEM:F12 3:1, 2% B27 supplement, MEM NEAA, 1X antibiotic, anti-mycotic and 1x GlutaMAX). On d25-30, optic vesicle-like structures were manually dissected and maintained as free floating organoids in poly HEMA (Sigma)-coated flasks with twice weekly feeding of 3D-RDM (RDM + 5% FBS (WiCell), 100 mM taurine (Sigma) and 1:1000 chemically defined lipid supplement) to which 1 mM all-trans retinoic acid (Sigma) was added until d100
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Organoids were dissociated to single cells with papain (Worthington) to 1 mg ml-1 and 5 μL DNase (Roche) per mL, using 200 μL papain mix per organoid. After 1-2 hours when organoids appeared fully dissociated the reaction was quenched with media containing 10% FBS (Gibco). Single cells were resuspended in HBSS (Gibco) and 0.1 mg mL-1 BSA at 120,000 cells per mL
Illumina nextera XT multiplexed with N70X indices according to the Dropseq protocol
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
single cell RNA
|
| Data processing |
Fastq files were processed and filtered according to the Dropseq cookbook version 1.0.1
processed reads were aligned to GRCh38 using the STAR RNA aligner
exons were tagged using the Homo_sapiens.GRCh38.88.gtf annotations file
Genome_build: GRCh38
Supplementary_files_format_and_content: raw UMI count matrices with genes as rows and cells as columns
|
| |
|
| Submission date |
Jan 14, 2020 |
| Last update date |
Oct 11, 2024 |
| Contact name |
Alyssa Kallman |
| E-mail(s) |
[email protected]
|
| Organization name |
Johns Hopkins University, School of Medicine
|
| Lab |
Donald Zack
|
| Street address |
400 N Broadway
|
| City |
Baltimore |
| State/province |
Maryland |
| ZIP/Postal code |
21231 |
| Country |
USA |
| |
|
| Platform ID |
GPL16791 |
| Series (1) |
| GSE143669 |
Investigating cone photoreceptor development using patient-derived NRLnull retinal organoids |
|
| Relations |
| BioSample |
SAMN13862728 |
| SRA |
SRX7556707 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4271891_L75P-9_d103-2.dge.txt.gz |
318.1 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
