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| Status |
Public on Nov 27, 2019 |
| Title |
RGC061616p7_A9_S297 |
| Sample type |
SRA |
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| Source name |
RGC061616p7_A9_S297
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| Organism |
Mus musculus |
| Characteristics |
mouse line: W3-GFP
age: postnatal day 15
tissue: retinas
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| Treatment protocol |
W3-GFP mice mice were used. Retinas were dissected in Ames solution (Sigma-Aldrich; equilibrated with 95% O2/5% CO2 for all use) immediately following enucleation.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Retinas were dissected in oxygenated AMES, digested in papain, and dissociated to single cell suspensions using manual trituration in ovomucoid solution. Single cell suspensions were incubated with 0.5ul of anti-CD90 (conjugated to various fluorophores) (Thermo Fisher Scientific) for 10 million cells per 100µl. Cells were sorted using a MoFlo Astrios. Cellular debris, doublets, and dead cells were excluded, and RGCs were collected based on high Cd90 and GFP co-expression. Singe cells were sorted into 2ul lysis buffer per well in multiple 96 well plates, spun down for 1min and immediated placed on dry ice, then stored at -80oC until subsequent processing for Smartseq2
We generated single cell RNA-Seq libraries using a modified Smart-seq2 method [Picelli et al., 2013, Ding et al., 2019] with the following minor change. We added 3 µl instead of 4 µl of master mix containing only 1.7 µl instead of 2.7 µl of 1 M Trehalose (Sigma-Aldrich) directly to the 2 µl cell lysate without a SPRI bead cleanup step.
Smart-seq2 per Picelli et al., 2013 with minor modifications (see above)
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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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)
Genome_build: mm10
Supplementary_files_format_and_content: Csv files including gene expression matrix from all samples
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| Submission date |
Sep 23, 2019 |
| Last update date |
Nov 27, 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 |
GPL19057 |
| Series (2) |
| GSE137400 |
Single-cell profiles of retinal neurons differing in resilience to injury reveal neuroprotective genes |
| GSE137863 |
Single-cell profiles of retinal neurons differing in resilience to injury reveal neuroprotective genes - Atlas of adult mouse retinal ganglion cells [Smart-seq2] |
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| Relations |
| BioSample |
SAMN12822018 |
| SRA |
SRX6889655 |
| 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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