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| Status |
Public on Oct 01, 2019 |
| Title |
CK |
| Sample type |
SRA |
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| Source name |
intestinal cells
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| Organism |
Danio rerio |
| Characteristics |
tissue: Intestinal single cell suspension
age: 16-weeks-old
exposed pollutants: none
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| Treatment protocol |
Exposure solutions were prepared by adding 100 nm, 5 μm or 200 μm PS-MPs to culture water with a final concentration of 500μg/L. The exposure solution were was replaced every 2 days. Exposure solution in all tanks were was continuously aerated to maintain the dispersion of particles (no filtering systems were used in the tanks). After 21-d exposure, zebrafish were collected and intestine were rapidly extracted on ice.
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| Extracted molecule |
total RNA |
| Extraction protocol |
The isolated intestinal tissue was digested into cell suspension with dispase. Cells were loaded on a GemCode Single Cell Instrument(10x Genomics, USA) to generate single cell Gel bead in Emulsion (GEMs).
ScRNA-seq libraries were prepared using the GemCode Single-Cell 3’ Gel Bead, Chip and Library Kits (10x Genomics, USA) as per the manufacturer’s protocol. Libraries were sequenced on an Illumina Hiseq PE150.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
We use FastQC to perform basic statistics on the quality of the raw reads. Then, those read sequences produced by the Illumina pipeline in FASTQ format were pre-processed through Trimmomatic software which can be summarized as below:(1) Remove low-quality reads: scan the read with a 4-base wide sliding window, cutting when the average quality per base drops below 10 (SLIDINGWINDOW: 4:10) (2) Remove trailing low quality or N bases (below quality 3) (TRAILING:3) (3) Remove adapters : there are two modes to remove the adapter sequence: a. alignment with the adapter sequence, the number of matching bases were greater than 7 and mismatch=2; b.when read1 and read2 overlapping base scoring greater than 30, removed non-overlapping portions (ILLUMINACLIP: adapter.fa: 2: 30: 7) (4) Drop reads below the 26 bases long (5) Discard those reads that can not form paired
The remaining reads that passed all the filtering steps was counted as clean reads and all subsequent analyses were based on this. At last, we use FastQC to perform basic statistics on the quality of the clean reads.
Cell Ranger uses an aligner called STAR, which peforms splicing-aware alignment of reads to the genome. Cell Ranger then uses the transcript annotation GTF to bucket the reads into exonic, intronic, and intergenic, and by whether the reads align (confidently) to the genome. A read is exonic if at least 50% of it intersects an exon, intronic if it is non-exonic and intersects an intron, and intergenic otherwise. For reads that align to a single exonic locus but also align to 1 or more non-exonic loci, the exonic locus is prioritized and the read is considered to be confidently mapped to the exonic locus with MAPQ 255. Cell Ranger further aligns exonic reads to annotated transcripts, looking for compatibility. A read that is compatible with the exons of an annotated transcript, and aligned to the same strand, is considered mapped to the transcriptome. If the read is compatible with a single gene annotation, it is considered uniquely (confidently) mapped to the transcriptome. Only reads that are confidently mapped to the transcriptome are used for UMI counting.
Cell Ranger takes as input the expected number of recovered cells, N (see -- expect-cells). Let m be a robust estimate of the maximum total UMI counts, taken as the 99th percentile of the top N barcodes by total UMI counts. All barcodes whose total UMI counts exceed m/10 are called as cells. This is performed separately for each GEM group (library) and, if the reference contains multiple genomes, for each genome.
Genome_build: Danio_rerio_Ensemble_91
Supplementary_files_format_and_content: gene-cell expression matrix
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| Submission date |
Aug 13, 2019 |
| Last update date |
Oct 02, 2019 |
| Contact name |
Gu Weiqing |
| E-mail(s) |
gwq960722@sina.com
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| Phone |
13962113111
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| Organization name |
Nanjing University
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| Street address |
Xianlin
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| City |
Nanjing |
| ZIP/Postal code |
210023 |
| Country |
China |
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| Platform ID |
GPL24995 |
| Series (2) |
| GSE135767 |
Single Cell RNA Sequencing Analysis of Polystyrene Microplastic Exposed and control Zebrafish Intestine |
| GSE136109 |
Single Cell RNA Sequencing and Intestinal Microbial Analysis of Polystyrene Microplastic Exposed Zebrafish Intestine |
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| Relations |
| BioSample |
SAMN12566347 |
| SRA |
SRX6707773 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4029392_CK.mtx.tsv.tar.gz |
8.9 Mb |
(ftp)(http) |
TAR |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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