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| Status |
Public on Dec 27, 2023 |
| Title |
Sublibrary_6 |
| Sample type |
SRA |
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| Source name |
Eye
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| Organism |
Gallus gallus |
| Characteristics |
tissue: Eye
treatment: Embryonic day 4, Embryonic day 5, Embryonic day 4 6 hours post-retinectomy, Embryonic day 4 6 hours post-retinectomy + FGF2
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| Treatment protocol |
Retinectomies and FGF2 delivery were performed by creating an incision around the anterior eye chamber with surgical scissors and the neural retina was dislodged with a wire probe. For FGF2-delivery, approximately 20 acrylic beads with immobilized surface heparin (MilliporeSigma, H5263) were incubated overnight in 4 µl of 250 ng/µl of bovine basic FGF2 (R&D Biosystems, 133-FB-025). At the time of surgery, FGF2 beads were deposited into the posterior chamber of the eye cup and the eye was closed. Embryos were incubated for 6 hours before enucleating the eyes for use with the Parse Biosciences snRNA-seq workflow.
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| Growth protocol |
Fertile Specific Pathogen Free eggs (Charles River Laboratories, 10100329) were incubated in a rotating, humidified incubator at 37° C.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Whole chicken eyes were enucleated at E4 or E5 in PBS containing 0.2 U / µl RNasin Plus RNase Inhibitor (Promega, N2615). Eyes from 3 embryos were pooled for each condition. Nuclei were immediately extracted and fixed for snRNA-seq. In brief, nuclei extraction was carried out via an optimized variation of the demonstrated protocols described in 10X Genomics Protocol CG000124 Rev E and 10X Genomics Protocol CG000366 Rev D. For snRNA-seq, cell lysis was performed by triturating sample and incubating in a cold buffer comprised of 10 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl2, 0.01% Surfact-Amps NP-40 (Thermo Fisher Scientific, 28324), and 0.2 U / µl RNase Inhibitor. Nuclei were washed 3 times by resuspending in cold PBS containing 2% fraction V bovine serum albumin and 0.2 U / µl RNase inhibitor (Promega, N2615). Nuclei were passed twice through 20 µm cell strainers (pluriSelect, 43-10020-40) during isolation to filter debris and cell aggregates.
snRNA-seq libraries were generated via the split-pool barcoding method using the Parse Biosciences Single Cell Whole Transcriptome Kit Chemistry Version 1 (Parse Biosciences, SB2001). Nuclei were fixed according to the Nuclei Fixation Kit Protocol (Parse Biosciences, SB1003) and stored at -80°C. During library preparation, nuclei were loaded for library prep as follows: embryonic day 4 (wells A1-A6), embryonic day 5 (wells A7-B1), embryonic day 4 6 hours post-retinectomy (wells B2-B8), and embryonic day 4 6 hours post-retnectomy + FGF2 (wells B9-C3). cDNA was amplified with 13 total PCR cycles. Final libraries were assessed with the Agilent Bioanalyzer and Qubit 4. Libraries were sequenced across two lanes of HiSeq X Ten and a lane of NovaSeq 6000 at the Novogene Sequencing Core (Sacramento, CA).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Reads were aligned to chicken genome GRCg7b and analyzed using the chicken annotation from Ensembl Release 109 using the Parse Biosciences alignment suite (v1.0.3)
Assembly: GRCg7b
Supplementary files format and content: all_genes.csv contains the list of genes output from the Parse Biosciences alignment suite
Supplementary files format and content: cell_metadata.csv contains the list of cell barcodes and gene counts output from the Parse Biosciences alignment suite
Supplementary files format and content: DGE.mtx contains the digital gene expression matrix output from the Parse Biosciences alignment suite
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| Submission date |
Jul 10, 2023 |
| Last update date |
Dec 27, 2023 |
| Contact name |
Katia Del Rio-Tsonis |
| E-mail(s) |
delriok@miamioh.edu
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| Organization name |
Miami University
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| Department |
Biology
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| Street address |
700 E High St
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| City |
Oxford |
| State/province |
OH |
| ZIP/Postal code |
45056 |
| Country |
USA |
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| Platform ID |
GPL26853 |
| Series (2) |
| GSE236902 |
Integrated single-cell multiomics uncovers foundational regulatory mechanisms of lens development and pathology [scRNA-seq] |
| GSE236905 |
Integrated single-cell multiomics uncovers foundational regulatory mechanisms of lens development and pathology. |
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| Relations |
| BioSample |
SAMN36379404 |
| SRA |
SRX20959227 |
| 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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