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Status |
Public on May 06, 2021 |
Title |
RNA-Seq E12_5 |
Sample type |
SRA |
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Source name |
mouse brain cortex
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Organism |
Mus musculus |
Characteristics |
tissue: somatosensory cortex developmental stage: E12.5 strain: C57BL/6NCrl genotype: wild type chromium single cell 3' solution chemistry: v2
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Treatment protocol |
For embryos E14.5 or younger we dissected the head, for older animals we isolated the brains. Dissection was performed in Hybernate E (Brainbits). The tissue was then embedded in 3% low melting agarose at 35-37C. Once the agarose solidified, the tissue was sectioned in 250um thick sections in a vibratome in iced Hybernate E. Sections were transferred to another plate and the prospective somatosensory cortex was dissected and meninges removed. For the earliest time points (E11.5 and E12.5) the prospective somatosensory cortex (medio-lateral region) was dissected without prior sectioning. Tissue was kept in cold buffers and on ice all time. RNAse free technique was used for handling. Cortical tissue from 4 animals was pulled together for each timepoint. For the Fezf2 experiments, samples were directly dissected from the cortex and processed individually, till genotype confirmation, when we combined samples from embryos with the same genotype. We genotyped embryos through PCR and qPCR on DNA extracted quickly from tail clips (QuickExtract DNA Extraction Solution, Lucigen), and through B-galactosidase detection assays.
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Extracted molecule |
polyA RNA |
Extraction protocol |
For single cell RNA seq, tissue pieces were processed to obtain single cell suspension using papain digestion (15-30 minutes according to embryo age) (Papain dissociation kit, Worthington). We followed the protocol as per the provider’s instructions. After dissociation and concentration, cells were resuspended in BSA 0.04% in PBS, at a concentration of 800-1200 cells/uliter. Cells were counted in a hemocytometer chamber and immediately processed for single cell GEM formation (10x Genomics, single cell RNA sequencing 3’, Chromium V2 or V3 for Fezf2 experiments). For single cell RNA seq we loaded the 10x chips aiming to recover 7,000–10,000 cells.cDNA amplification and library construction were done following 10xGenomics instructions. For the complete wild type developmental atlas we generated Chromium V2 libraries, while Chromium V3 was used for all Fezf2 experiments. Libraries were quantified in BioAnalyzer and sequenced in HiSeq or NovaSeq illumina platforms. single cell RNA sequencing 3’, Chromium V2 or V3
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina HiSeq 2000 |
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Data processing |
Raw sequencing data was processed via the Cell Ranger pipeline (10xGenomics, version 2.0.1) to generate a count matrix from the bcl files. Genome_build: mm10 (GRCm38.84) Supplementary_files_format_and_content: Unfiltered feature-barcode matrices HDF5
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Submission date |
Jun 24, 2020 |
Last update date |
May 08, 2021 |
Contact name |
Paola Arlotta |
E-mail(s) |
paola_arlotta@harvard.edu
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Organization name |
Harvard University
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Department |
Stem Cell and Regenerative Biology
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Lab |
Arlotta Lab
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Street address |
7 Divinity Ave
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City |
Cambridge |
State/province |
MA |
ZIP/Postal code |
02138 |
Country |
USA |
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Platform ID |
GPL13112 |
Series (2) |
GSE153162 |
Molecular Logic of Cellular Diversification in the Mouse Cerebral Cortex (scRNAseq) |
GSE153164 |
Molecular Logic of Cellular Diversification in the Mouse Cerebral Cortex |
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Relations |
BioSample |
SAMN15357672 |
SRA |
SRX8609911 |
Supplementary file |
Size |
Download |
File type/resource |
GSM4635073_E12_5_filtered_gene_bc_matrices_h5.h5 |
39.4 Mb |
(ftp)(http) |
H5 |
SRA Run Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
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