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| Status |
Public on May 04, 2023 |
| Title |
scRNA-seq - Control |
| Sample type |
SRA |
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| Source name |
Retina
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| Organism |
Mus musculus |
| Characteristics |
cell type: Muller glia
mouse line: GlastCreER;R26R-EYFP
strain: C57BL/6
condition: mCherry
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| Treatment protocol |
Animals were electroporated with a Cre-dependent control construct, received tamoxifen at P21 or older, and were sacrificed 3 weeks post-tamoxifen.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Electroporated retina pieces were dissociated in papain (Worthington). YFP+ cells from both mCherry+ (n=4 retinas) or mCherry- (n=5 retinas) regions were pooled. Fluorescent cells were sorted with a FACSAria III Cell Sorter (BD Biosciences). Viable YFP+ cells (single cells) were collected in PBS with 0.15% BSA (Millipore). Cells were then spun at 300g for 10 minutes at 4°C and re-suspended in 0.15% BSA/PBS.
Cells were loaded on a 10xGenomics Single Cell 3’ chip and processed according to manufacturer V3 pipeline 3.1.0 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 |
Scanpy.loom files containing count matrix, features and barcodes. .h5 files containing cellranger or cellranger-arc output count matrix, features and barcodes whereas .tsv file containing aligned fragment coordinates.
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| Data processing |
scRNA-seq sequencing data were aligned to the mm10 genome using CellRanger version 6.1.2 (Cell Ranger software, 10x Genomics,). Filtered output files were analyzed in Python (Python core team, Python) using Scanpy version 1.9.1 (Wolf et al., 2018). Genes detected in less than 3 cells were removed from the analysis and low-quality cells (less than 200 genes detected, more than 2500 genes detected or more than 5% of mitochondrial genes) were removed from the datasets. To assign individual cell types, scDeepSort version 1.0 (Shao et al., 2021) was used alongside with deep learning trained model grounded on previously published retinal single cell expression data (Clark et al., 2019). Full YFP and mCherry sequences were reconstructed using RNA-Bloom version 1.4.3 (Nip et al., 2020) and Trinity version 2.14.0 (Grabherr et al., 2011), and then remapped to the initial sequencing data. Doublets were identified using Scrublet version 0.2.2 (Wolock et al., 2019) and removed before downstream analysis. Batch correction, data integration, gene expression plotting, differential expression data and further analysis were performed using scVI-tools version 0.19.0 (Gayoso et al., 2022).
Raw ATAC-seq fastq reads were aligned using bowtie2 mm9 genome on Galaxy platform. MACS2 was used for peak calling using these parameters: --nomodel --shift -37 --extsize 73 . Bamcoverage was used to generate bigwig files.
Salmon quant function was used to quantify effective length of transcripts and transcript per million value from RNA-seq raw fastq reads using mm10 transcriptome. Salmon quants output files were used to calculate Log2FC and statistical values using DESeq2 for differential analysis. Annotate DESeq2 package on Galaxy platform was used to annotate gene_ids to symbols.
Genome_build: mm10, mm9
Supplementary_files_format_and_content: Bigwig tracks files for ATAC-seq conditions and bed peak files for scored peaks.
Supplementary_files_format_and_content: Matrix table with Log2FC and FDR values of comparisons between the two conditions.
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| Submission date |
Mar 24, 2021 |
| Last update date |
May 21, 2023 |
| Contact name |
Awais Javed |
| E-mail(s) |
awaisj14@gmail.com, awais.javed@ircm.qc.ca
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| Phone |
5149875772
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| Organization name |
IRCM
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| Lab |
Michel Cayouette
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| Street address |
110 Avenue des Pins
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| City |
Montreal |
| State/province |
Quebec |
| ZIP/Postal code |
H2W1R7 |
| Country |
Canada |
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| Platform ID |
GPL24247 |
| Series (1) |
| GSE169519 |
Direct neuronal reprogramming by temporal identity factors |
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| Relations |
| BioSample |
SAMN18474480 |
| SRA |
SRX10435060 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5208239_Control_filtered_feature_bc_matrix.h5 |
9.4 Mb |
(ftp)(http) |
H5 |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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