|
| Status |
Public on Feb 22, 2023 |
| Title |
scRNAseq-mm-Wt-HL-E115-Rep1-L20666 |
| Sample type |
SRA |
| |
|
| Source name |
Hindlimb
|
| Organism |
Mus musculus |
| Characteristics |
tissue: Hindlimb
genotype: Wild Type
embryonic stage: E11.5
strain: c57bl6.J
|
| Treatment protocol |
Limbs from E11.5 mouse embryos were dissected in 1xPBS. A single-cell suspension was obtained by incubating the tissue for 10 min at 37 °C in 200 µL Gibco trypsin-EDTA 0.05% (Thermo Fisher Scientific) and 20 µL 5% BSA (Sigma-Aldrich). Digestion was then blocked by adding 400 µL of BSA 5%. Cells were then resuspended by pipetting and filtered using a 0.40 µm cell strainer (Falcon). Cells were washed once with 0.04% BSA with a centrifugation step performed for 5 min at 1200 r.p.m then resuspended in 0.04% BSA.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The cell concentration was determined using an automated cell counter (Bio-Rad) and cells were subjected to single-cell RNA-sequencing (10x Genomic, ChromiumTM Single Cell 3’ v2; one reaction per time point and per strain has been used) aiming for a target cell recovery of up to 10,000 sequenced cells per sequencing library (time point and strain). Single-cell libraries were generated according to the manufacturer’s instructions with the following conditions: 8 PCR cycles were ran during cDNA amplification and 12 PCR cycles were ran during library generation/sample indexing to increase library complexity. Libraries were sequenced with a minimum of 230 million paired end reads.
scRNA-seq 10X Genomics
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina MiSeq |
| |
|
| Description |
scRNAseq-matrix-counts.mtx.gz
scRNAseq-genes_ids.tsv.gz
scRNAseq-cellmetadata.tsv.gz
scRNAseq-barcodes.tsv.gz
|
| Data processing |
The Cell Ranger pipeline v.3 (10x Genomics lnc.) was used for each scRNA-seq sample in order to de-multiplex the raw base call files, to generate the fastq files, and to perform the alignment against a custom mouse reference genome mm9 to create the unique molecular identifier (UMI) count matrix.
Only cells with more than 2000 detected genes and less than 10% of mitochondrial UMI counts were considered for further analysis.
We used scrublet (https://doi.org/10.1016/j.cels.2018.11.005) to identify potential duplets in our dataset. We filtered out cells with a scrublet score higher than 0.2.
Each sample dataset was normalized independently using the SCT method (10.1186/s13059-019-1874-1) implemented in Seurat3 (10.1038/nbt.4096) R package and then integrated the datasets using the Seurat3 IntegrateData function and considering the top 2000 most variable genes.
The first 20 Principal Components of this joint dataset were calculated and used for UMAP projection and cell clustering. To delimitate the major hindlimb bud cell types, we used the Louvain algorithm implemented in the Seurat3 function FindClusters and the expression of well-known marker genes for the limb-comprising cell types. For the AER cluster, we used the marker gene Fgf8. Differential gene expression between the WT and the Dup cells was estimated by modelling the gene expression as a function of the genotype across cells. We fitted a quasi-Poisson distribution to calculate the effect of the Dup genotype on the gene expression distribution of each gene using the monocle3 strategy. We tested that such effect was not equal to 0. We use this condition association effect to rank the genes and identified the pathways associated with the top 200 genes using the Enrichr tool from the Maayan lab50. Finally, we confirmed that the top perturbed genes associated to the muscle pathways are more frequently expressed in the Dup AER cells.
Assembly: mm10
|
| |
|
| Submission date |
Jan 22, 2023 |
| Last update date |
Feb 22, 2023 |
| Contact name |
Giulia Cova |
| E-mail(s) |
Giulia.cova@nyulangone.org
|
| Organization name |
Max Planck Institute
|
| Street address |
63-73 Ihnestrasse
|
| City |
Berlin |
| ZIP/Postal code |
14195 |
| Country |
Germany |
| |
|
| Platform ID |
GPL16417 |
| Series (2) |
| GSE197404 |
Combinatorial effects on gene expression at the Lbx1/Fgf8 locus resolve Split-Hand/Foot Malformation type 3 |
| GSE223456 |
Combinatorial effects on gene expression at the Lbx1/Fgf8 locus resolve Split-Hand/Foot Malformation type 3 [scRNA-seq] |
|
| Relations |
| BioSample |
SAMN32868426 |
| SRA |
SRX19136133 |
