run_metadata: 32366
This data as json
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| 32366 | SRR29152187 | SRX24673329 | SRS21408057 | SRP509548 | PRJNA1115803 | Stem and progenitor cell proliferation are independently regulated by cell type specific cyclinD genes | GSE268255 | Transcriptome Analysis | Regeneration and homeostatic turnover of solid tissues depend on the proliferation of symmetrically dividing adult stem cells which either remain stem cells or differentiate based on their niche position. Here we demonstrate that in zebrafish lateral line sensory organs stem and progenitor cell proliferation are independently regulated by two cyclinD genes. Loss of ccnd2a impairs stem cell proliferation during development while loss of ccndx disrupts hair cell progenitor proliferation but allows normal differentiation. Notably ccnd2a can functionally replace ccndx indicating that the respective effects of these Cyclins on proliferation are due to cell type specific expression. However even though hair cell progenitors differentiate normally in ccndx mutants they are mispolarized due to hes2 and Emx2 downregulation. Thus regulated proliferation ensures that equal numbers of hair cells are polarized in opposite directions. Our study reveals cell type specific roles for cyclinD genes in regulating the different populations of symmetrically dividing cells governing organ development and regeneration with implications for regenerative medicine and disease. Overall design: Two replicates per sibling or ccndx mutant were used | ccndx sibling2 | GSM8288756 | tissue:5dpf zebrafish FACS sorted lateral line cells|cell type:Homostasis|genotype:Wildtype|geo loc name:missing|collection date:missing | ccndx sibling2 | Raw sequencing data were processed using 10X Genomics Cell Ranger pipeline. Reads were demultiplexed into Fastq file format using cellranger mkfastq. Genome index was built by cellranger mkref using zebrafish genome danRer11 ensembl 98 gene model. Data were aligned by STAR aligner and cell counts tables were generated using cellranger count function with default parameters. Assembly: danRer11 Supplementary files format and content: single cell count matrix in hdf5 format raw data in bam format. | 5dpf zebrafish FACS sorted lateral line cells | All cells were collected during homeostasis no treatments were performed. | 5 dpf zebrafish were anesthetized with Tricane then split between two 70mm cell strainers Falcon and placed in 0.25% Trypsin EDTA Gibco on ice. The larvae from each strainer are then added to a 5ml round bottom tube Falcon and the tissue is broken up via slowly pipetting up and down using a pipette man and a glass pipette VWR keeping the tubes on ice. The total time from placing larvae in trypsin to finishing pipetting is approximately 8 minutes. The tissue is then filtered through 70mm filter BD Biosciences pelleted for 5 min at 2000rpm at 4oC and then the pellet is resuspended in 1xDPBS/0.04%BSA. The pelleting and resuspension in 1xDPBS/0.04%BSA is repeated two more times. post the final pellet the cells are resuspended in 0.5mL 1xDPBS/0.04%BSA then cells are combined and put through another 70mm filter into a 5ml round bottom tube. Cells are then incubated in Draq5 1/200 dilution for 5 minutes on ice. Draq5+/mCherry+ cells were sorted by the Stowers Institute Cytometry Core Facility using a BD Influx Cell Sorter BD Biosciences San Jose CA. USA with cells collected in tubes containing 10mL 1XDPBS/0.04% BSA. Cells were not fixed in methanol as in previous protocols as we found this gave inconsistent results with 10X Genomics version 3 kits. Sorted cells are then spun down at 350rcf at 4oC for 2 min in a swinging bucket centrifuge with slow deceleration. Most of the supernatant is removed and the cells are resuspended in approximately remaining 100mL. Cells deemed to be at least 80% viable were loaded based on live cell concentration onto a Chromium X Series instrument 10x Genomics and libraries prepared using the Chromium Next GEM Single Cell three prime Reagent Kits v3.1 10x Genomics according to manufacturer’s directions. Resulting cDNA and short fragment libraries were checked for quality and quantity using a 2100 Bioanalyzer Agilent Technologies and Qubit Fluorometer Thermo Fisher Scientific. With cells captured estimated at 6 800 9 600 cells per sample libraries were pooled and sequenced to a depth necessary to achieve at least 30 000 mean reads per cell on an Illumina NextSeq 2000 instrument utilizing RTA and instrument software versions current at the time of processing with the following paired read lengths: 28*10*10*90bp. scRNA seq 10x Genomics | 5dpf zebrafish FACS sorted lateral line cells were used. Standard growing condition were used. | cell type:Homostasis|genotype:Wildtype | GSM8288756 | GSM8288756: ccndx sibling2; Danio rerio; RNA Seq | GSM8288756 r1 | GSM8288756 | 1 | 5 dpf zebrafish were anesthetized with Tricane then split between two 70mm cell strainers Falcon and placed in 0.25% Trypsin EDTA Gibco on ice. The larvae from each strainer are then added to a 5ml round bottom tube Falcon and the tissue is broken up via slowly pipetting up and down using a pipette man and a glass pipette VWR keeping the tubes on ice. The total time from placing larvae in trypsin to finishing pipetting is approximately 8 minutes. The tissue is then filtered through 70mm filter BD Biosciences pelleted for 5 min at 2000rpm at 4oC and then the pellet is resuspended in 1xDPBS/0.04%BSA. The pelleting and resuspension in 1xDPBS/0.04%BSA is repeated two more times. post the final pellet the cells are resuspended in 0.5mL 1xDPBS/0.04%BSA then cells are combined and put through another 70mm filter into a 5ml round bottom tube. Cells are then incubated in Draq5 1/200 dilution for 5 minutes on ice. Draq5+/mCherry+ cells were sorted by the Stowers Institute Cytometry Core Facility using a BD Influx Cell Sorter BD Biosciences San Jose CA. USA with cells collected in tubes containing 10mL 1XDPBS/0.04% BSA. Cells were not fixed in methanol as in previous protocols as we found this gave inconsistent results with 10X Genomics version 3 kits. Sorted cells are then spun down at 350rcf at 4oC for 2 min in a swinging bucket centrifuge with slow deceleration. Most of the supernatant is removed and the cells are resuspended in approximately remaining 100mL. Cells deemed to be at least 80% viable were loaded based on live cell concentration onto a Chromium X Series instrument 10x Genomics and libraries prepared using the Chromium Next GEM Single Cell three prime Reagent Kits v3.1 10x Genomics according to manufacturer's directions. Resulting cDNA and short fragment libraries were checked for quality and quantity using a 2100 Bioanalyzer Agilent Technologies and Qubit Fluorometer Thermo Fisher Scientific. With cells captured estimated at 6 800 9 600 cells per sample libraries were pooled and sequenced to a depth necessary to achieve at least 30 000 mean reads per cell on an Illumina NextSeq 2000 instrument utilizing RTA and instrument software versions current at the time of processing with the following paired read lengths: 28*10*10*90bp. scRNA seq 10x Genomics | RNA-Seq | TRANSCRIPTOMIC SINGLE CELL | cDNA | PAIRED | ILLUMINA | NextSeq 2000 | SRP509548 | assembly:danRer11|intentional duplicate | ccndx_sibling2.bam | 10X Genomics bam file | 27438063300.0 | 304867370.0 | GSM8288756 r1 | 0:90 | A:7768441003;C:5988780076;G:6567352571;T:7111630965;N:1858685 | 90 | 7768441003 | 5988780076 | 6567352571 | 7111630965 | 1858685 | SRX24673329 | SRS21408057 | SRA1876130 | Computational Biology, Stowers Institute for Medical Research | Computational Biology, Stowers Institute for Medical Research | 1 | 0.94768 | 0.20814 | 0.75452 | 0.52756 | 90 | B | usable mapping rate | illumina | nextseq_v2 | unknown | cdna_unspecified | unknown | sc | single_cell_droplet | 10x | United States | 2024-05-23 | Larval | Larval | Lateral Line | Sensory System |