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Sample GSM4996498 Query DataSets for GSM4996498
Status Public on Feb 04, 2021
Title In vivo grafted hESC-RPE and rabbit, rabbit 2
Sample type SRA
 
Source name In vitro hESC-derived culture grafted into albino rabbit retina, sorted
Organisms Homo sapiens; Oryctolagus cuniculus
Characteristics tissue/cell type: In vitro hESC-derived culture grafted into albino rabbit retina, sorted
Extracted molecule total RNA
Extraction protocol A rabbit eye with 30-day integrated hESC-RPE were enucleated and pigmented areas including neuroretina, choroid and RPE layer was dissected out, trimmed and mixed together in 500 uL of digestion buffer. Digestion buffer consisted of: 2mg/ml collagenase IV (ThermoFisher Scientific, 17104019), 120 U/μl DNase I (NEB, Sigma, 4536282001), and 1mg/ml papain (Sigma, 10108014001) in PBS. Eppendorfs containing the samples were rotated and incubated at 37°C on a thermocycler at 300g for 25 min until samples were homogenized. Samples were pipetted every 5 min to digest the tissue sample into single cells. Digestion was stopped by adding equivalent volume of 10% fetal bovine serum (ThermoFisher Scientific, 10082147) in PBS, the samples were filtered using a 30µm MACS Smart Strainer (Miltenyi, 130-098-458) followed by Dead Cell Removal kit (Miltenyi, 130-090-101) to remove dead cells and debris. The sample was stained with mouse anti-human HLA-ABC-FITC (1:20, BD Biosciences, 555552, clone [G46-2.6]), and anti-human HLA-ABC-positive cells were FACS-sorted as specified above, collected and resuspended to 1000 cells/uL in 1% BSA (Sigma, A1470-10g) in PBS for further scRNA-sequencing.
Cells were transported at 4°C to the Eukaryotic Single Cell Genomics Facility (ESCG, SciLifeLab, Stockholm, Sweden) where a 3’ cDNA library was prepared for single cell RNA sequencing (scRNA-seq) using the 10X Genomics platform and NovaSeq 6000 software.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NovaSeq 6000
 
Description P15064_1001_S1
Data processing Cell Ranger 2.1.1 was used to convert Illumina base call files to FASTQ format.
Cell Ranger 3.1.0 was used to map FASTQ sequencing reads to the human GRCh38 reference transcriptome with the STAR aligner and to generate feature-barcode count matrices.
For cells isolated from rabbit retina were generated using a custom-built hybrid reference transcriptome combining both the human GRCh38 reference and the rabbit reference (Oryctolagus cuniculus 2.0.99, EMBL-EBI). FASTQ files were mapped to this hybrid reference with Cell Ranger 3.1.0 to generate count matrices. Reads were exclusively assigned either to a human or rabbit transcript.
The velocyto run10x command was used to produce loom files containing spliced and unspliced RNA count matrices
Downstream analyses, including dimensionality reduction and cell type annotation, were performed using both velocyto and scanpy in Python (version 3.7)
Genome_build: GRCh38 human reference, Oryctolagus cuniculus 2.0.99 EMBL-EBI rabbit reference
Supplementary_files_format_and_content: Processed data were stored in loom file format (http://loompy.org/) containing spliced and unspliced count matrices. For the human-rabbit cell mixture (rabbit1.tar.gz, rabbit2.tar.gz), processed data is provided as the 10X count matrices in mtx file format).
 
Submission date Dec 31, 2020
Last update date Feb 04, 2021
Contact name Gioele La Manno
E-mail(s) gioele.lamanno@epfl.ch
Organization name EPFL
Department BMI
Lab DSBL
Street address Station 19
City Lausanne
State/province Vaud
ZIP/Postal code 1015
Country Switzerland
 
Platform ID GPL29549
Series (1)
GSE164092 Molecular profiling of stem cell-derived retinal pigment epithelial cell differentiation established for clinical translation
Relations
BioSample SAMN17190581
SRA SRX9763499

Supplementary file Size Download File type/resource
GSM4996498_rabbit2.tsv.mtx.tar.gz 8.0 Mb (ftp)(http) TAR
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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