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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Apr 01, 2024 |
| Title |
SC22 - Periosteum and hematoma, tibia 3 days post-fracture |
| Sample type |
SRA |
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| Source name |
Periosteum
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| Organism |
Mus musculus |
| Characteristics |
tissue: Periosteum
genotype: C57BL6
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| Extracted molecule |
total RNA |
| Extraction protocol |
For uninjured periosteum, tibias from 4 mice were dissected free of muscle and surrounding tissues. The epiphyses were cut and the bone marrow flushed. The periosteum was scraped from the cortex using Dissecting Chisel (10095-12, Fine Science Tools). For day 5 and 7 post-fracture, injured tibias from 4 to 9 mice were collected and the surrounded tissues were removed. The activated periosteum was scraped and collected with the hematoma. Collected tissues were minced and placed 5 min in ice-cold Nuclei Buffer (NUC101, Merck) before mechanical nuclei extraction using a glass douncer. Extraction was performed by 20 strokes of pestle A followed by 10-20 of pestle B. Nuclei suspension was filtered, centrifuged and resuspended in RNAse-free PBS (AM9624, ThermoFischer Scientific) with 2% Bovine Serum Albumin (A2153, Merck) and 0.2 U/µL RNAse inhibitor (3335399001, Roche). A second step of centrifugation was performed to reduce contamination by cytoplasmic RNAs. Sytox™ AADvanced™ (S10349, ThermoFischer Scientific) was added (1/200) to label nuclei and Sytox-AAD+ nuclei were sorted using Sony SH800.
The snRNA-seq libraries were generated using Chromium Single Cell Next GEM 3′ Library & Gel Bead Kit v.3.1 (10x Genomics) according to the manufacturer’s protocol. Briefly, 10 000 to 20 000 nuclei were loaded in the 10x Chromium Controller to generate single-nuclei gel- beads in emulsion. After reverse transcription, gel-beads in emulsion were disrupted. Barcoded complementary DNA was isolated and amplified by PCR. Following fragmentation, end repair and A-tailing, sample indexes were added during index PCR. The purified libraries were sequenced on a Novaseq (Illumina) with 28 cycles of read 1, 8 cycles of i7 index and 91 cycles of read 2. Sequencing data were processed using the Cell Ranger Count pipeline and reads were mapped on the HG38 2020-A reference human genome, with intronic and exonic sequences.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
10x Genomics
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| Data processing |
Fastq files were processed with the CellRanger pipeline with the default’s parameters
Reads were align against the mm10 reference genome
Filtered features barcodes matrices were used to perform data analysis
Assembly: mm10
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| Submission date |
Jun 08, 2023 |
| Last update date |
Apr 01, 2024 |
| Contact name |
Céline Colnot |
| E-mail(s) |
celine.colnot@inserm.fr
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| Organization name |
INSERM U955
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| Lab |
Colnot Lab
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| Street address |
8 rue du Général Sarrail
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| City |
Créteil |
| ZIP/Postal code |
94000 |
| Country |
France |
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| Platform ID |
GPL24247 |
| Series (1) |
| GSE234451 |
Single nuclei RNAseq of the periosteal response to bone fracture in mice |
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| Relations |
| BioSample |
SAMN35675636 |
| SRA |
SRX20639361 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7468836_SC22_barcodes.tsv.gz |
4.3 Kb |
(ftp)(http) |
TSV |
| GSM7468836_SC22_features.tsv.gz |
284.1 Kb |
(ftp)(http) |
TSV |
| GSM7468836_SC22_matrix.mtx.gz |
3.6 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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