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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Nov 01, 2022 |
| Title |
E12.5 |
| Sample type |
SRA |
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| Source name |
Molar at embryonic day 12.5
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
tissue: molar
timepoint: day 12.5
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| Growth protocol |
All animal experiments were approved by the Institutional Animal Care and Use Committee at College of Life Sciences, Sichuan University. All animals were maintained under standardized conditions with the temperature- and light-controlled (12 hr light/dark cycle), in individually ventilated cages always with companion mice and had free access to food and water. Knock-in transgenic mice (Pitx2-P2A-copGFP and Msx1-P2A-tdTomato) were generated by Biocytogen, Inc (Beijing).
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| Extracted molecule |
total RNA |
| Extraction protocol |
The molar tooth germs were physically separated from the mandibular using dissection needles under a stereo-fluorescent microscope. Tissues surrounding the tooth germs were eliminated and tooth germs were collected into a clean culture plate. Next, the tooth germs were washed twice with D-PBS and incubated with 0.8 ml 0.75 mg/ml Dispase in 1% FBS D-PBS for 20-45 min (E12.5 20 min; E14.5 30 min; E16.5 35 min; PN1 40 min; PN7 45 min). After that, 3 ml culture medium supplied with 20 µl 5 mg/ml DNase was used to neutralize the enzymes and prevent cell aggregates. The epithelium and mesenchyme were then separated and dissociated with 0.25% Trypsin-EDTA for 5 min and 9 min respectively in an incubator at 37°C with 5% CO2. Finally, single-cell suspensions were collected through a 70 μm strainer and centrifuged at 550 g for 5 min. Cell number and viability were assessed by an automatic cell counter.
Using single cell 3’ Library and Gel Bead Kit V3 (10x Genomics) and Chromium Single Cell B Chip Kit (10x Genomics), the cell suspension (300-600 living cells per micro-liter determined by Count Star) was loaded onto the Chromium single cell controller (10x Genomics) to generate single-cell gel beads in the emulsion according to the manufacturer’s protocol. In short, single cells were suspended in PBS containing 0.04% BSA. About 6,000 cells were added to each channel, and the target cell recovered was estimated to be about 3,000 cells. Captured cells were lysed and the released RNA were barcoded through reverse transcription in individual GEMs. Reverse transcription was performed on a S1000TM Touch Thermal Cycler (Bio Rad) at 53℃ for 45 min, followed by 85℃ for 5 min, and hold at 4℃. The cDNA was generated and then amplified, and quality assessed using an Agilent 4200. According to the manufacture’s introduction, Single-cell RNA-seq libraries were constructed using Single Cell 3’ Library and Gel Bead Kit V3. The libraries were finally sequenced using an Illumina HiSeq X or MiSeq sequencer with a sequencing depth of at least 100,000 reads per cell with pair-end 150 bp (PE150) reading strategy.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
HiSeq X Ten |
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| Data processing |
The Cell Ranger (v3.0.2) software was obtained from 10x Genomics website (https://support.10xgenomics.com/single-cell-gene-expression/software/downloads/latest). Alignment, filtering, barcode counting, and UMI counting were performed with cellranger count module to generate feature-barcode matrix and determine clusters.
Genome_build: mm10
Supplementary_files_format_and_content: feature_bc_matrix
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| Submission date |
Dec 01, 2020 |
| Last update date |
Nov 02, 2022 |
| Contact name |
Zhonghan Li |
| E-mail(s) |
zhonghan.li@scu.edu.cn
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| Phone |
+86-28-85463752
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| Organization name |
Sichuan University
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| Department |
College of Life Science
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| Street address |
29 Wangjiang Road
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| City |
Chengdu |
| State/province |
Sichuan |
| ZIP/Postal code |
610064 |
| Country |
China |
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| Platform ID |
GPL21273 |
| Series (1) |
| GSE162413 |
The cellular basis for epithelial-mesenchymal interaction during mouse tooth development |
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| Relations |
| BioSample |
SAMN16968422 |
| SRA |
SRX9611888 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4951212_E12.5-filtered_feature_bc_matrix.tar.gz |
89.8 Mb |
(ftp)(http) |
TAR |
| GSM4951212_E12.5-filtered_feature_bc_matrix.zip |
89.8 Mb |
(ftp)(http) |
ZIP |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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