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| Status |
Public on Oct 21, 2021 |
| Title |
Mouse_Brain_1 |
| Sample type |
SRA |
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| Source name |
Mouse brain cortex/hippocampus
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| Organism |
Mus musculus |
| Characteristics |
tissue: Brain
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. C57BL/6J mice from Jackson Laboratories were used for mouse experiments. 50 uL of pooled AAV vector library was delivered by systemic injections via facial vein injection to P0 mice, which were anesthetized on ice. Tissues were collected 3 weeks 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. Brain, heart, and liver of mice were freshly dissected, and cells were dissociated using Macs Miltenyi Adult Brain Tissue Dissociation Kit (130-107-677), Multi Tissue Dissociation Kit 2 (130-110-203) and Liver Dissociation Kit (130-105-807) according to manufacturer’s recommendations. The cells were resuspended in 0.1% BSA in D-PBS and processed immediately for scRNA-seq. Following dissociation using Macs Miltenyi Tissue Dissociation Kits specific for retina, brain, heart and liver, a Miltenyi MACS Tyto sorter was used to enrich for GFP-positive cells. Cells were resuspended 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. Samples from mouse tissues and cultured 293AAV (Cell Biolabs) cells were prepared for single cell analysis using a 10X Chromium Single Cell 3’ v3.1 kit. The resulting libraries were pooled, and an additional targeted gene enrichment protocol was performed using 10X Chromium Targeted Gene Expression kit.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
mouse_barcodes.tsv
mouse_genes.tsv
aav_barcode_key_mouse.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.
The performance of AAV variants was analyzed based on quantification of AAV variant-mediated GFP-barcode mRNA expression. For non-human primates, AAV-barcodes were analyzed from (1) the original scRNA-seq data and (2) PCR amplification of GFP from the 10x single cell prepped sample library. Mouse samples were analyzed using AAV- barcodes from (1) the original scRNA-seq data and (2) targeted gene enrichment against GFP and other marker genes. Targeted gene enrichment samples from the mouse were downsampled to a similar number of reads as the non-human primate GFP PCR-amplified non-human primate samples. AAV-barcodes were identified using Salmon (v0.9.1) transcript quantification. Only reads with 1 hit to an AAV- barcode were kept. Using these reads, AAV variants were identified based on the AAV- barcode. 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. As each UMI (unique molecular identifier) represents a single mRNA molecule captured, only one AAV- barcode should exist for each UMI. Rarely, multiple AAV- barcodes were found per UMI – possibly due to sequencing/PCR-introduced errors – in which 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. Mouse samples were aligned to the GRCm38 reference GCA_000001635.5 from NCBI.
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 |
Oct 20, 2021 |
| Last update date |
Oct 22, 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 |
GPL24247 |
| Series (1) |
| GSE161645 |
scAAVengr, a transcriptome-based pipeline for quantitative ranking of engineered AAVs with single-cell resolution |
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| Relations |
| BioSample |
SAMN22426696 |
| SRA |
SRX12705135 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5641048_B1_raw_matrix.mtx.gz |
142.2 Mb |
(ftp)(http) |
MTX |
| GSM5641048_cell_by_aav_mouse_B1_brain_results.csv.gz |
4.7 Kb |
(ftp)(http) |
CSV |
| GSM5641048_mouse_barcodes.tsv.gz |
17.5 Mb |
(ftp)(http) |
TSV |
| GSM5641048_mouse_genes.tsv.gz |
439.3 Kb |
(ftp)(http) |
TSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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