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| Status |
Public on Feb 06, 2019 |
| Title |
M2PerCD90S1 |
| Sample type |
SRA |
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| Source name |
Macaque 6 retina
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| Organism |
Macaca fascicularis |
| Characteristics |
tissue: Peripheral retina
sorting: CD90 enrichment
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| Treatment protocol |
Eyes from male and female macaques (Macaca fascicularis) of 3-9 years of age were generously provided by Biomere (Worcester, MA), Mikhail Papisov,Curtis Cetrulo, or Tatsuo Kawai (Massachusetts General Hospital) or purchased from Toxikon Corporation (Bedford, MA). For sequencing and labeling, macaque eyes were collected either under deep anesthesia or 15-45 min postmortem, then immediately stored as either whole globes in ice-cold Ames solution (Sigma-Aldrich; equilibrated with 95% O2/5% CO2 for all use) or as posterior poles in room temperature Ames solution following a rapid hemisection to remove the vitreous and the anterior chamber. Retinas were dissected free from the posterior pole, following hemisection in the case whole globes, and then stored in ice-cold Ames solution. Experiments described below commenced within 8 hours (depending on travel time from provider sites).
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| Extracted molecule |
total RNA |
| Extraction protocol |
Peripheral retinal pieces were dissected and pooled from all quadrants of the retina. Single cell suspensions were dissociated as described for fovea. Dissociated cells were stained incubated with CD90 microbeads (Miltenyi Biotec, 130-096-253) to enrich RGCs or with anti-CD73 (BD Biosciences, clone AD2) followed by anti-mouse IgG1 microbeads (Miltenyi Biotec, 130-047-102) to deplete rods. CD90 positive cells or CD73 negative cells were selected via large cell columns through a MiniMACS Separator (Miltenyi Biotec). Single cell suspensions were diluted at a concentration of 500-1800 cells/mL in 0.04% BSA/Ames for loading into 10X Chromium Single Cell A Chips.
Single cell libraries were made with Chromium 3’ v2 platform (10X Genomics, Pleasanton, CA) following the manufacturer’s protocol. Briefly, single cells were partitioned into Gel bead in EMulsion (GEMs) in the GemCode instrument with cell lysis and barcoded reverse transcription of RNA, followed by amplification, shearing and 5’ adaptor and sample index attachment. On average, approximately 10,000 single cells were loaded on each channel and approximately 6,000 cells were recovered. Libraries were sequenced on Illumina HiSeq 2500.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
CountMatrix_M6perCD90.csv
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| Data processing |
Sample demultiplexing with the mkfastq function from Cell Ranger software(version 2.1.0, 10X Genomics)
Sample alignment with the count function from Cell Ranger software(version 2.1.0, 10X Genomics), we used the “--force-cells 6000” option to get a “loose” upper bound on the number of cells that could be recovered
Genome_build: We used a custom assembled transcriptome based on NCBI transcriptome for M. fascicularis (annotation release 101)
Supplementary_files_format_and_content: Csv files including gene expression matrix from each macaque by different sorting methods
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| Submission date |
Aug 21, 2018 |
| Last update date |
Feb 06, 2019 |
| Contact name |
Wenjun Yan |
| E-mail(s) |
wey334@g.harvard.edu
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| Organization name |
Harvard University
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| Department |
Department of Molecular and Cellular Biology
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| Lab |
Joshua Sanes
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| Street address |
52 Oxford Street
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| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02138 |
| Country |
USA |
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| Platform ID |
GPL20302 |
| Series (2) |
| GSE118480 |
Molecular specification of cell types underlying central and peripheral vision in primates |
| GSE118852 |
Molecular specification of cell types underlying central and peripheral vision in primates (macaque peripheral single cell RNA-seq) |
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| Relations |
| BioSample |
SAMN09873008 |
| SRA |
SRX4589924 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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