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| Status |
Public on Aug 01, 2020 |
| Title |
PEC_WT_2 |
| Sample type |
SRA |
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| Source name |
Pancreatic endocrine cells (PECs)
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| Organism |
Danio rerio |
| Characteristics |
strain: AB
tissue: Endocrine pancreas
age: 27 hpf
genotype: Wild type
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| Treatment protocol |
The following zebrafish transgenic and mutant lines were used: Tg(pax6b:GFP)ulg515 (Delporte et al., 2008b), TgBAC(pdx1:EGFP)bns13 (Helker et al., 2019), TG(-8.5nkx2.2a:GFP)ia2 (Pauls et al., 2007), pax6bsa0086 and pax6bsunrise (Verbruggen et al., 2010).
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| Growth protocol |
Zebrafish (Danio rerio) were raised according to standard protocols and staged according to Kimmel (Kimmel et al., 1995).
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| Extracted molecule |
total RNA |
| Extraction protocol |
Enteroendocrine cells (EECs) were isolated by dissecting the gut from about 200 Tg(pax6b:GFP)ulg515 larvae at 4 dpf, taking care of not including pancreatic tissue. Cell dissociation was next performed by incubation in HBSS 1x supplemented with 100 U/ml collagenase IV and 0.3 U/ml Dispase (Life Technologies) for 10 minutes. Cells were washed in HBSS (Mg2+ and Ca2+ free) containing 1% BSA and GFP-expressing EECs were selected by two consecutive FACS purifications, the first in the “yield mode” and the second in “the purity mode”, using FACS Aria II. Four replicates of EEC containing about 3000 cells were prepared. Pancreatic endocrine cells (PECs) were also obtained from the Tg(pax6b:GFP)ulg515 line (Delporte et al., 2008b) by dissecting the dorsal pancreatic bud from about 200 27-hpf transgenic embryos. FACS selection was performed as described for EECs except that cell dissociation was performed in Tryple Select 1X ( Gibco) supplemented with 100 U/ml collagenase IV (Life Technologies) for 5 minutes. For the preparations of EECs and PECs from pax6b null mutant embryos, the pax6sa0086 line (Verbruggen et al., 2010) was first crossed with the Tg(pax6b:GFP)ulg515 line; heterozygous pax6bsa0086 fish harboring the transgene (pax6b:GFP) were inbred to generate homozygous pax6bsa0086 transgenic embryos which were selected based on the absence or reduction of lens. The isolation of EECs and PECs from pax6bsa0086 homozygotes were performed in triplicates following the same procedure than for the wild-type larvae. The accuracy of pax6bsa0086 homozygotes selection was verified after the RNA-seq by checking the presence of the null sa0086 allele in 100% of pax6b reads in the mutant samples.
Each EEC or PEC sample obtained after FACS was directly pelleted by centrifugation and resuspended in 3.5 µl of reaction buffer, lysed by freezing in liquid nitrogen and stored at -80°C according the the Smart-seq2 protocol (Picelli et al., 2014). cDNA was synthesised and amplified by a 13 cycles PCR reaction. Quality of cDNA was verified by 2100 High Sensitivity DNA assay (Agilent technologies). 1 ng cDNA was used for preparing each cDNA library using Nextera-XT kit (Illumina) and sequenced on Hi-seq 2000 to obtain around 60 millions of reads (100 base paired-ends).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
Sequences were trimmed in order to remove adaptors and low quality bases
Trimmed reads were mapped in to the zebrafish genome GRCz11 using STAR software v.2.5.4b (Dobin et al., 2013) and gene expression was measured from the mapped reads by using built-in STAR module (--quantMode GeneCounts).
Genome_build: GRCz11 - release 92 (Ensembl)
Supplementary_files_format_and_content: tab-delimited text files include raw counts for annotations for each Sample
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| Submission date |
Apr 21, 2020 |
| Last update date |
Aug 01, 2020 |
| Contact name |
Arnaud Lavergne |
| Organization name |
University of Liège
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| Department |
GIGA
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| Street address |
Avenue de l'hôpital 11
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| City |
Liège |
| State/province |
Liège |
| ZIP/Postal code |
4000 |
| Country |
Belgium |
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| Platform ID |
GPL14875 |
| Series (1) |
| GSE149081 |
Pancreatic and intestinal endocrine cells share common transcriptomic signatures and gene regulatory networks |
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| Relations |
| BioSample |
SAMN14657539 |
| SRA |
SRX8152986 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4490213_PEC_WT_2.counts.txt.gz |
130.7 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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