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| Status |
Public on Nov 01, 2023 |
| Title |
P5 cortex |
| Sample type |
SRA |
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| Source name |
Brain
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| Organism |
Mus musculus |
| Characteristics |
cell type: Neurons
tissue: Brain
strain: Sst-Cre; RCEloxP
age: P5
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| Extracted molecule |
total RNA |
| Extraction protocol |
The neocortex of E16.5, P1, and P5 SstCre/+;RCEfl/+ mice was dissected in an ice-cold HBSS buffer supplement with 25 mM of glucose. For E16.5, tissue from 6 animals was pooled together. For P1 and P5, tissue from 3 animals was pooled together. Tissues were cut into small pieces and digested for 15 min at 37°C with carbogen oxygenation using 10ml of pronase dissociation buffer solution, pH7.5, Osmolarity 300-315, with the following composition: 0.2 mg/ml PronaseE (Sigma), 50mM of Trehalose, 20 mM glucose, 0.8 mM kynurenic acid, 0.05 mM APV, 0.09M Na2SO4, 0.03M K2SO4 and 0.014M MgCl2. For P5 tissue, tissue was incubated for 10 mins with dissociation buffer containing pronase, after which 500ul of 2.5% trypsin and 200ul of 10mg/ml DNAseI were added and incubated at 37°C for 2 more minutes. Post enzymatic digestion, tissues were washed once in ice-cold dissociation buffer without enzyme and the tissue was mechanically triturated into single cells in 2 ml of OptiMEM (Invitrogen) supplement with 2% Trehalose, 20 mM glucose, 0.8 mM kynurenic acid and 0.05 mM APV using 3 glass fire polished pipettes with decreasing diameter. For P1 and P5 tissue, we removed myelin by overlaying single cell suspension on top of a 30% percoll and centrifuge at 700 g for 10 mins. The cell pellet was resuspended in 300-500 µl of OptiMEM with 2% Trehalose, 20mM glucose, 0.8 mM kynurenic acid and 0.05 mM APV and tissue chunks were eliminated by passing cells suspension through a 40 µm cell strainer (BD). GFP+ and DAPI negative cells were isolated using fluorescence-activated cell sorting (FACS) with a flow cytometer (Becton Dickinson) using a 100 µm nozzle. Cells were sorted in bulk. In all experiments, we only proceed toward scRNAseq with > 80% viability. About 4000-6000 isolated single cells for each sample were added to a 10X Genomic single-cell machine for single-cell capture and cDNA library preparation. RNA-seq was performed in an Illumina high-seq 2500 platform.
Library was performed according to the manufacter’s instructions (single cell 3’ v3 protocol, 10x Genomics). Briefly, GCs were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
10x Genomics
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| Data processing |
Sequencing data were prepared for analysis by application of the Cell Ranger pipeline. First, Illumina BCL output files were de-multiplexed into FASTQ format files. Feature counts were computed for individual GEM wells. STAR aligner was applied to perform splicing aware alignment to the GRCm38 reference genome, and then reads were bucketed into exonic, intronic and intergenic categories. Reads were classified as confidently aligned if they corresponded to a single gene annotation. Only confidently aligned reads were carried forward to UMI counting. A cell calling algorithm in conjunction with the barcode rank plot was applied to filter low RNA content cells and remove empty droplets. The output is a read count matrix. The R package Seurat was used to performing the bulk of our subsequent data analysis, including filtering, normalisation, scaling and other downstream processing.
Assembly: GRCm38
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| Submission date |
Nov 01, 2022 |
| Last update date |
Nov 01, 2023 |
| Contact name |
jingyu wang |
| E-mail(s) |
jywang.rd@gmail.com
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| Organization name |
THU
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| Street address |
xueyuan street
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| City |
Beijing |
| ZIP/Postal code |
100083 |
| Country |
China |
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| Platform ID |
GPL17021 |
| Series (1) |
| GSE217065 |
Cortical somatostatin long-range projection neurons and interneurons exhibit divergent developmental trajectories |
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| Relations |
| BioSample |
SAMN31516285 |
| SRA |
SRX18074663 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6704387_P5barcodes.tsv.gz |
18.1 Kb |
(ftp)(http) |
TSV |
| GSM6704387_P5features.tsv.gz |
245.0 Kb |
(ftp)(http) |
TSV |
| GSM6704387_P5matrix.mtx.gz |
23.4 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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