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| Status |
Public on Oct 19, 2021 |
| Title |
Cynomolgus_Macaque_Sample12R_PCR_GFP |
| Sample type |
SRA |
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| Source name |
Cynomolgus_Macaque_Sample12R_PCR_GFP
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| Organism |
Macaca fascicularis |
| Characteristics |
tissue: Retina
region: Retina periphery region
sorting strategy: unsorted
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| Treatment protocol |
Marmosets, cynomolgus macaques and rhesus macaques between 3-10 years and retinas were injected intravitreally with AAV libraries. Monkeys used for K912-scCAG-GFP fluorophore expression received daily oral doses of cyclosporine at a dose of 6 mg/kg for the duration of the study. Marmosets received oral daily doses of meloxicam (0.2 mg/kg) for one week after injection.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
The NHP retinas were dissected and regions of interest were isolated (macula, superior and inferior periphery). For cynomolgus macaque, superior and inferior periphery were pooled. Retinal tissue was placed in Hibernate solution (Hibernate A -Ca Solution, BrainBits LLC), and cells were then dissociated using Macs Miltenyi Biotec Neural Tissue Dissociation Kit for postnatal neurons (130-094-802) according to manufacturer’s recommendations. Dissected retina pieces were incubated with agitation at 37 °C and further mechanically dissociated. The dissociated neural retina was filtered using a 70 µm MACS Smart Strainer (Miltenyi Biotec) to ensure single-cell suspension. Cells were resuspended in 0.1% BSA in D-PBS and processed immediately for scRNA-seq.
Marmoset and cynomolgus macaque samples were prepared for single cell analysis using a 10x Chromium Single Cell 3’ v3 kit. Briefly, single cells from retina samples were captured using 10X Chromium system (10X Genomics), the cells were partitioned into Gel beads-in-emulsion (GEMS), mRNAs were reverse transcribed and cDNAs with 10X Genomics Barcodes were created with unique molecular identifiers (UMIs) for different transcripts. Purified cDNA was PCR amplified and further purified with SPRIselect reagent (Beckman Coulter, B23318). Final libraries were generated after fragmentation, end repair, A-tailing, adaptor ligation, and sample index PCR steps according to 10x Single Cell 3’ workflow. An additional targeted sequencing analysis was run on these 10x-prepped cDNA samples, using PCR amplification with Q5 High Fidelity DNA Polymerase to target the GFP sequence and its associated AAV barcode.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina MiSeq |
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| Description |
PCR_GFP_Sample_12R_S16_L001
PCR-amplified 10X-processed reads, targeting GFP
supplementary or processed data file:
aav_barcode_key.txt
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| Data processing |
Sequencing data was demultiplexed into sample-level fastq files using Cell Ranger mkfastq (v3 10X Genomics).
Alignment and cell demultiplexing were run using STARsolo(14) (v2.7) with default parameters, creating raw count matrices.
GFP-barcodes were analyzed from the original scRNA-seq samples and PCR amplification of GFP from the 10x single cell prepped sample libraries. In both cases, the GFP-barcodes were identified using Salmon (Patro et al., 2017) (v0.9.1) transcript quantification. Only reads with 1 hit to a GFP barcode were kept. Using these reads, AAV variants were identified based on the GFP barcode (aav_barcode_key.txt). 10x barcodes in the reads from the PCR amplification analysis were corrected according to the 10x Cell Ranger count algorithm to mitigate any errors that may have been introduced by multiple rounds of PCR. Rarely, multiple AAV variants were found per UMI (unique molecular identifier), and in this case the AAV variant with the highest number of counts for that UMI was kept.
Genome_build: Cynomolgus macaque samples were aligned to the Macaca_fascicularis_5.0/macFas5 reference obtained from UCSC and marmoset samples were aligned to ASM275486v1 obtained from Ensembl.
Supplementary_files_format_and_content: Matrix tables with raw gene expression counts for every cell; Cell-by-GFP tables with raw GFP-barcode expression counts
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| Submission date |
Nov 17, 2020 |
| Last update date |
Oct 19, 2021 |
| Contact name |
Leah Byrne |
| E-mail(s) |
lbyrne@pitt.edu
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| Organization name |
University of Pittsburgh
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| Department |
Ophthalmology
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| Lab |
Byrne Lab
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| Street address |
3501 Fifth Avenue
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| City |
Pittsburgh |
| State/province |
PA |
| ZIP/Postal code |
15213 |
| Country |
USA |
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| Platform ID |
GPL19647 |
| Series (1) |
| GSE161645 |
scAAVengr, a transcriptome-based pipeline for quantitative ranking of engineered AAVs with single-cell resolution |
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| Relations |
| BioSample |
SAMN16821891 |
| SRA |
SRX9522463 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4911882_PCR_GFP_cell_by_aav_cyno_macaque_sample12R_results.csv.gz |
26.7 Kb |
(ftp)(http) |
CSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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