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| Status |
Public on Jan 22, 2020 |
| Title |
Single-nuclei RNAseq (NucSeq) of human eyes |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
11 samples total with libraries prepared on 10X Chromium. Corresponding to 4 control eyes.
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| Overall design |
**Raw Data not provided due to patient privacy concerns**
Methods: Donor eyes with no retinal disease diagnosis from the Lions Eye Institute (Tampa, FL) were enucleated and flash frozen in LN2 within 5 h postmortem or less, shipped on dry ice and stored at -80C. The eye globes were cryosectioned at 60 μm starting at the base of the optic nerve and at a plane perpendicular to the optic nerve. Posterior sections with visible RPE layers were collected in batches of 8 for NucSeq (see below). Additional sections of more anterior eye regions were used to assess overall RNA quality. 108955 (right eye) is from a 78 year old female donor, PMI = 3:40 hours, RNA integrity number (RIN) = 8.0; 120974 (left eye) is from a 79 year old male donor, PMI = 2:40 hours, RIN = 8.0; 109829 (left eye) is from a 77 year old female donor, PMI = 4:08 hours, RIN = 8.4; 109373 (right eye) is from a 70 year old male donor, PMI = 4:40 hours RIN = 8.6; 110814 (right eye) is from a 65 year old male donor, PMI = 4:25 hours, RIN = 8.5. For donor eye 108955, sections 1-8 (region 1, most poterior) and 9-18 (region 2) are separately subjected to nuclei extraction and library construction with the 10X Chromium single cell 3’ gene expression kit v2. For the remaining 4 donor eyes, only region 2 was used for NucSeq with the v3 kit. Nuclei were isolated and processed based on methods in (Krishnaswami et al., 2016). Briefly, sections were thawed directly in ice cold homogenization buffer (250 mM sucrose, 25 mM KCl, 5 mM MgCl2, 10 mM Tris buffer pH 8.0, 1 μM DTT, 1X cOmpleteTM protease inhibitors with EDTA (Roche), 0.1% v/v Triton X 100, 0.4U/μL recombinant RNAse Inhibitor (Ambion), 0.2U/μL SUPERas-In (Ambion), 0.2 μg/ml DAPI) and dounced 15X with a loose pestle in a glass tissue homogenizer (Wheaton). Nuclei released were washed once in homogenization buffer and sorted for 2N DNA content gated on DAPI (linear scale) on a FACS-ARIA II (BD) with cooled chamber. Sorted single nuclei were collected in 1% BSA/1X PBS with 0.2U/μL RNase Inhibitor, concentrated, resuspended in ~50 μL residual buffer, counted manually with a hemocytometer, and immediately processed with 10X Chromium single cell 3’ gene expression kit v2 or v3 (10X Genomics) at ~16000 nuclei per library and 2-3 libraries per region, following manufacturer’s protocol.
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| Contributor(s) |
Orozco LD, Chen H |
| Citation(s) |
31995762, 37691820 |
| Submission date |
Jul 30, 2019 |
| Last update date |
Sep 15, 2023 |
| Contact name |
Luz D Orozco |
| E-mail(s) |
orozcogl@gene.com
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| Organization name |
Genentech Inc.
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| Department |
Bioinformatics
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| Lab |
Orozco Lab
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| Street address |
1 DNA Way
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| City |
South San Francisco |
| State/province |
CA |
| ZIP/Postal code |
94080 |
| Country |
USA |
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| Platforms (1) |
| GPL16791 |
Illumina HiSeq 2500 (Homo sapiens) |
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| Samples (11)
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| Relations |
| BioProject |
PRJNA557496 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE135133_RAW.tar |
316.4 Mb |
(http)(custom) |
TAR (of TXT) |
| GSE135133_clusterAssignments.txt.gz |
536.9 Kb |
(ftp)(http) |
TXT |
| Raw data not provided for this record |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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