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Sample GSM7840723 Query DataSets for GSM7840723
Status Public on Nov 30, 2023
Title 0, 1, 3, 12 hpa regeneration
Sample type SRA
 
Source name tail
Organism Xenopus laevis
Characteristics tissue: tail
treatment: amputated
Treatment protocol Single cell experiment was performed using 0, 1, 3, 12 hpa of Xenopus tail at stage 42. To avoid artificial activation of gene expression and keep cells at a low temperature, the process of the whole tissue dissociation was done in a refrigerated room at 4 °C. Embryos were anesthetized and the regenerating part was dissected and collected into 0.5 ml of 2/3 PBS (Sigma D8537, diluted by RNase-free water) with Actinomycin D (ActD, 50 μg/ml; Sigma A1410, storage solution 5 mg/ml in DMSO). Tail pieces from 50 embryos were collected per one sample.
Growth protocol Xenopus laevis females were stimulated with 500 U of human chorionic gonadotropin (Sigma-Aldrich). Eggs were collected the following day and fertilized by testes suspension which were surgically obtained from the male. After the removal of jelly coats by the 2% cysteine treatment, embryos were incubated in 0.1x MBS until the experimental procedure. Embryonic stages were determined based on Nieuwkoop and Faber table. Embryos were immediately transferred to the 0.1x MMR solution with gentamycin. Solution was changed every day.
Extracted molecule polyA RNA
Extraction protocol Tissue was resuspended in 200 μl of dissociation solution I containing papain , BSA (40 μg/ml,), ActD (50 μg/ml), protease (0.5 mg/ml) and DNase I (50 μg/ml). Dissociation was gently resuspended using P200 wide-bore tip for one minute and gently rotated for 2 minutes. After three repeated resuspending/rotating steps, samples were shortly spun down, supernatant with cells were collected into tubes with 1 ml of fetal bovine serum (FBS, Gibco) prechilled on ice. Undissociated pieces were resuspended in another 200 μl of dissociation solution I and dissociated for another three steps of resuspending and rotating. Supernatant with cells was collected into a new tube with 1 ml of FBS. Next, the tissue was resuspended in 200 μl of dissociation solution II (papain with CaCl2 (5 mM), BSA (40 μg/ml), ActD (50 μg/ml), protease (5 mg/ml) and DNase I (50 μg/ml)) and dissociation was performed for three rounds of 10 minutes dissociation the same as described above. In total, five tubes with single-cell suspension in FBS were collected per sample. This allowed for both the preservation of the quality of sensitive cells (released first) and also the collection of the inner cells (collected last).
Sequencing libraries were prepared according to the manufacturer’s manual “Chromium Single Cell 3' Reagent Kits User Guide (v 3.1)”.
In total 2400 cells per sample were loaded into the Chromium chip. Library quality was tested by capillary electrophoresis on Fragment Analyzer (Agilent, NGS High Sensitivity kit, DNF-474). The sample libraries were then pooled and sequenced on Illumina NovaSeq 2000 targeting 100000 read pairs per cell.
 
Library strategy RNA-Seq
Library source transcriptomic single cell
Library selection cDNA
Instrument model Illumina NovaSeq 6000
 
Description 10x Genomics
Data processing Data were processed using STAR v2.7.9a. Reads were aligned and counted against Xenopus laevis genome (v 10.1) with annotation XENLA_10.1_GCF obtained from Xenbase
Raw unfiltered data were processed using R packages. Firstly, droplets with cells were selected using DropletUtils v1.14.1 using command emptyDrops with parameter ”lower = 1000” and FDR <= 0.001. Filtered matrix were used for next processing using Seurat v4.1.0.
Assembly: Xenopus laevis genome (v 10.1)
Supplementary files format and content: barcodes.tsv: raw cell barcodes
Supplementary files format and content: features.tsv: raw list of gene IDs
Supplementary files format and content: matrix.mtx: raw gene expression count data in Matrix Market Exchange Format
Supplementary files format and content: processed_metadata.xlsx: metadata of the filtered and processed data, including the time points and cell type annotations
 
Submission date Oct 13, 2023
Last update date Nov 30, 2023
Contact name Radek Sindelka
E-mail(s) Radek.Sindelka@ibt.cas.cz
Organization name Institute of Biotechnology, Czech Academy of Science, v.v.i.
Lab Laboratory of Gene Expression
Street address Průmyslová 595
City Vestec by Prague
ZIP/Postal code 25250
Country Czech Republic
 
Platform ID GPL28901
Series (2)
GSE245312 Characterization of regeneration initiating cells during Xenopus laevis tail regeneration [scRNA-Seq]
GSE245320 Characterization of regeneration initiating cells during Xenopus laevis tail regeneration
Relations
BioSample SAMN37810362
SRA SRX22090550

Supplementary file Size Download File type/resource
GSM7840723_barcodes.tsv.gz 17.5 Mb (ftp)(http) TSV
GSM7840723_features.tsv.gz 300.7 Kb (ftp)(http) TSV
GSM7840723_matrix.mtx.gz 122.8 Mb (ftp)(http) MTX
GSM7840723_processed_metadata.xlsx 302.3 Kb (ftp)(http) XLSX
SRA Run SelectorHelp
Raw data are available in SRA

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