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| Status |
Public on Dec 15, 2020 |
| Title |
Forelimb bud at embryonic day E10.5 |
| Sample type |
SRA |
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| Source name |
Forelimb bud
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| Organism |
Mus musculus |
| Characteristics |
genotype: Hoxa13:Cre/+;mT-mG
tissue: Forelimb bud
strain: C57BL/6X129
developmental stage: embryonic day E10.5
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Dissection of forelimb buds from Hoxa13:Cre/+;mT-mG embryos were performed at E10.5, E11.5 and E12.5. Forelimbs were dissected in cold 1XPBS, after centrifugation at 300 rcf for 5 min at 4 °C the forelimbs were incubated in dissociation buffer (450 μl of 0.25% Trypsine/EDTA (GIBCO), 50 μl 10% bovine serum albumin (BSA), 1 μl DNAseI (NEB)) for 10 min at 37 °C. After incubation limb cells were gently mixed by pipetting up and down 10–15 times until they were dissociated, then 10% final fetal bovine serum (FBS) was added. Dissociated cells were filtered using a cell strainer (40 μm Nylon, BD Falcon), counted in order to determine the final volume in which to resuspend the cells and assessed for cell viability using 0.4% Trypan blue. After centrifugation at 300 rcf for 7 min at 4 °C the resulting pellet of cells was resuspended in the appropriate volume of 1XPBS, 0.04% BSA in order to have a 1500 cells/μl. Before being processed dissociated cells were counted one more time and assessed for cell viability using 0.4% Trypan blue.
The single cell preparation was processed using Chromium Single Cell 3' Reagent Kits v3 (10X Genomics, Pleasanton, CA) following the manufacturer recommendation. Briefly, forelimb cells were partitioned into gel beads in emulsion for cell lysis, barcoding with oligo-dT primers and reverse transcription in order to produce barcoded, full-length cDNA from poly-adenylated mRNA. cDNA library was amplified, fragmented and size selected. Samples were controlled at multiple steps during the procedure by running on BioAnalyzer.
10X Chromium Single Cell 3`. Libraries were sequenced on NovaSeq 6000 with 100 bp paired-end reads.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
Forelimb bud at embryonic day E10.5
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| Data processing |
sample demultiplexing using Cell Ranger Single-Cell Software Suite version3.0.1
Sequencing reads were aligned on a custom genome on which sequences of IRES-venus, tdTomato, Cre et eGFP were added to Mus_musculus.GRCm38.93. Genome. using Cell Ranger Single-Cell Software Suite version3.0.1
filtering using Cell Ranger Single-Cell Software Suite version3.0.1
barcode counting using Cell Ranger Single-Cell Software Suite version3.0.1
UMI counting using Cell Ranger Single-Cell Software Suite version3.0.1
Genome_build: Sequencing reads were aligned on a custom genome on which sequences of IRES-venus, tdTomato, Cre et eGFP were added to Mus_musculus.GRCm38.93. Genome.
Supplementary_files_format_and_content: datasets created from the out files generated by the cellranger count function (Cell Ranger Single-Cell Software Suite version3.0.1) using Read10X function from Seurat R package v3.1
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| Submission date |
Sep 30, 2020 |
| Last update date |
Dec 15, 2020 |
| Contact name |
Marie Kmita |
| E-mail(s) |
marie.kmita@ircm.qc.ca
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| Organization name |
IRCM (Institut de Recherches Cliniques de Montréal)
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| Street address |
110 Pine Avenue
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| City |
Montreal |
| State/province |
QC |
| ZIP/Postal code |
H2W 1R7 |
| Country |
Canada |
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| Platform ID |
GPL24247 |
| Series (1) |
| GSE158820 |
Transcriptional trajectories highlight the transition from anterior-posterior to proximal-distal patterning in the early limb bud [scRNA-seq] |
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| Relations |
| BioSample |
SAMN16306868 |
| SRA |
SRX9220413 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4811354_E105.data.R.gz |
62.0 Mb |
(ftp)(http) |
R |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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