 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Nov 27, 2019 |
| Title |
RGC061616p9_E9_S249 |
| Sample type |
SRA |
| |
|
| Source name |
RGC061616p9_E9_S249
|
| Organism |
Mus musculus |
| Characteristics |
mouse line: W3-GFP
age: postnatal day 15
tissue: retinas
|
| 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.
|
| 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)
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| 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
|
| |
|
| Submission date |
Sep 23, 2019 |
| Last update date |
Nov 27, 2019 |
| Contact name |
Wenjun Yan |
| E-mail(s) |
wey334@g.harvard.edu
|
| Organization name |
Harvard University
|
| Department |
Department of Molecular and Cellular Biology
|
| Lab |
Joshua Sanes
|
| Street address |
52 Oxford Street
|
| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02138 |
| Country |
USA |
| |
|
| 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] |
|
| Relations |
| BioSample |
SAMN12821915 |
| SRA |
SRX6889752 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
|
|
|
|
 |
