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| Status |
Public on Sep 27, 2019 |
| Title |
NoHCC [Single-cell] |
| Sample type |
SRA |
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| Source name |
Liver
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| Organism |
Danio rerio |
| Characteristics |
transgenic line: Tg(fabp10a-flox-beta-catenin)
age: 6 months post fertilization
treatment at larval stage: 10micromolar 4-hydroxytamoxifen from 3 to 6 days post fertilization.
diagnosis: No HCC
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| Treatment protocol |
Larvae for Samples 2 and 3 were treated with 10micromolar 4-hydroxytamoxifen from 3 to 6 days post fertilization.
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| Growth protocol |
Fish were raised following IACUC-approved protocols in insititutional zebrafish facility.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Zebrafish were euthanized by rapid chilling and their livers were dissected. Half of each liver was submitted for histologic evaluation to confirm the diagnosis (HCC or no HCC). The remaining half of each liver was dissociated into single-cell suspensions and prepared for single-tube single-cell RNA sequencing (scRNA-seq) based on the 10X Genomics platform. Dissected liver tissue was immersed in 5% Fetal Bovine Serum (FBS) in Hank’s Buffered Saline Solution (HBSS) without phenol red, with calcium and magnesium and chopped finely. The cells were were homogenized in 1 mL of 0.25% trypsin + EDTA for 5 minutes at room temperature and re-suspended in 1 mL of 5%FBS+5mM EDTA in HBSS-Free solution. Cells were then filtered through a 40 micron membrane filter, and the filtrate was centrifuged at 1200 RPM, 4C for 5 minutes. The cell pellet was then re-suspended in phosphate buffered saline with 0.04% bovine serum albumin. The cell suspension was then filtered again through 40 micron cell strainers to obtain a liver single-cell suspension.
Viability and cell count were assessed on Countess I (Thermo Scientific). The Chromium Single Cell Gene Expression Solution with 3’ chemistry, version 2 (PN-120237) was used to barcode individual cells with 16 bp 10x Barcode and to tag cell specific transcript molecules with 10 bp Unique Molecular Identifier (UMI) according to the manufactures instruction. Equilibrium to targeted cell recovery of 6,000 cells along with 10x Gel Beads and reverse transcription reagents were loaded to Chromium Single Cell A Chip (PN-120236) to form Gel-Bead-In EMulsions (GEMs), the micro-droplets. Within individual GEMs, cDNA generated from captured and barcoded mRNA was synthesized by reverse transcription at the setting of 53°C for 45 min followed by 85°C for 5 min. Subsequent A tailing, end repair, adaptor ligation and sample indexing was performed in bulk according to the manufacturer's instructions. Multiple libraries were then normalized and sequenced on NovaSeq 6000 with 2x150 PE mode.
Single-cell 3' RNA-Sequencing
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
Tg(fabp10a-flox-β-catenin)
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| Data processing |
10x Genomics’ Cell Ranger software (v2.2.0) executed primary data analysis for each sample. Pipeline 'cellranger mkfastq' generated R1 (26 bp) and R2 (100 bp) FASTQ files.
Custom transgenic genomic reference was built with ‘cellranger mkref’, but shared the common Danio rerio genome reference build GRCz11 with annotations from Ensembl release 94. The Ensembl gene annotations were filtered with ‘cellranger mkgtf’ for gene_biotypes matching ‘protein_coding’, ‘lincRNA’ and ‘antisense’ tags. All samples had additional transgenic sequence/annotations added.
Each sample was processed with ‘cellranger count’ pipeline with their respective transgenic genome build with parameter ‘--expect-cells=3000’
In attempt to recover those (perhaps lower quality) GEM partitions, the raw gene-barcode matrices from ‘cellranger count’ (located in ‘outs/raw_gene_bc_matrices’) was processed with the EmptyDrops algorithm (R package DropletUtils v1.2.2) to discriminate cells from background GEM partitions at a false discovery rate (FDR) of 1% (Lun ATL et al., 2019). GEM partitions with 500 UMI counts or less were considered to be devoid of viable cells, while those with at least 10,000 UMI counts were automatically considered to be cells.
Cell-based QC metrics were calculated with R package scater (v1.10.1) using the calculateQCMetrics function (McCarthy DJ et al., 2017). Principal component analysis (PCA) on the cell-based QC metrics combined with a multivariate outlier method flagged cells with outlying values in QC metrics as suspect (P. Filzmoser et al., 2008). Cells with extremely low UMI counts, extremely low gene counts or extremely high percentage of expression attributed to mitochondrial genes were also flagged as low quality. Extremeness in any of these three measures was determined by 3 median absolute deviations from the median with the scater::isOutlier function applied to each sample individually. Additionally, cells were required to have greater than 800 UMIs and less than 20% of total expression attributed to mitochondrial transcripts. Those cells suspected of being low quality were removed from downstream analysis.
Genome_build: GRCz11
Supplementary_files_format_and_content: MTX
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| Submission date |
Sep 20, 2019 |
| Last update date |
Sep 28, 2019 |
| Contact name |
Kimberley Jane Evason |
| Organization name |
University of Utah
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| Department |
Pathology
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| Street address |
2000 Circle of Hope
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| City |
Salt Lake City |
| State/province |
UT |
| ZIP/Postal code |
84112 |
| Country |
USA |
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| Platform ID |
GPL18413 |
| Series (2) |
| GSE137784 |
Single-cell transcriptomic analysis of two models of zebrafish beta-catenin-driven HCC |
| GSE137788 |
Transcriptomic analysis of two models of zebrafish beta-catenin-driven HCC |
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| Relations |
| BioSample |
SAMN12797705 |
| SRA |
SRX6878621 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4087821_15547X4_barcodes.tsv.gz |
5.1 Kb |
(ftp)(http) |
TSV |
| GSM4087821_15547X4_genes.tsv.gz |
80.6 Kb |
(ftp)(http) |
TSV |
| GSM4087821_15547X4_matrix.mtx.gz |
2.7 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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