|
| Status |
Public on May 02, 2020 |
| Title |
KK-S6 |
| Sample type |
SRA |
| |
|
| Source name |
embryo_head
|
| Organism |
Danio rerio |
| Characteristics |
sample type: embryo_head_2dpf
developmental stage: 2 day post-fertilization
genotype: fam50a KO
|
| Growth protocol |
Embryos were obtained from natural matings of adult fam50a heterozygous KO mutants zebrafish that were maintained on a 14h/10h light/dark cycle. Embryos were reared in embryo media (0.3 g/L NaCl, 75 mg/L CaSO4, 37.5 mg/L NaHC03, 0.003% methylene blue) at 28°C until 2 dpf.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
We extracted total RNA from embryo heads with Trizol (ThermoFisher), according to manufacturer's instructions. N=20 embryos/pool. Samples were treated with DNase prior to RNAseq
RNA-seq libraries were prepared using the commercially available KAPA Stranded mRNA-Seq Kit following the manufacturer’s protocol. In brief, mRNA transcripts were first captured using magnetic oligo-dT beads, fragmented using heat and magnesium, and reverse transcribed using random priming. During the 2nd strand synthesis, the cDNA:RNA hybrid was converted into double-stranded cDNA (dscDNA) and dUTP incorporated into the 2nd cDNA strand. Illumina sequencing adapters were ligated to the dscDNA fragments and amplified to produce final RNA-seq libraries. Libraries were indexed and pooled in an equimolar ratio prior to 50 bp single end sequencing on the same lane of a HiSeq 4000 (Illumina). Sequence data were demultiplexed and Fastq files were generated using Bcl2Fastq conversion software (Illumina).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Data processing |
RNA-seq data were processed using the TrimGalore toolkit (http://www.bioinformatics.babraham.ac.uk/projects/trim_galore) which employs Cutadapt to trim low-quality bases and Illumina sequencing adapters from the 3’ end of reads. Only reads that were 20 nt or longer after trimming were kept for further analysis. Reads were mapped to the Zv10r87 version of the zebrafish genome and transcriptome using the STAR RNA-seq alignment tool and retained for subsequent analysis if they mapped to a single genomic location. Gene counts were compiled using the HTSeq tool (http://www-huber.embl.de/users/anders/HTSeq/), and only genes that had ≥10 reads in any given library were used in subsequent analyses.
Normalization and differential expression were carried out using the DESeq2 Bioconductor package with the R statistical programming environment (www.r-project.org). False discovery rate was calculated to control for multiple hypothesis testing.
Gene set enrichment analysis was performed to identify gene ontology terms associated with altered gene expression for each comparison.
Alternative splicing was characterized using the rMATS algorithm, which employs the STAR alignment tool along with the Zv10r87 version of the zebrafish genome and transcriptome.
DAVID pathway analysis was run on significant genes (FDR <= 5%) on each of the five alternative splicing event categories as well as the union set of all five lists.
Genome_build: Zv10r87 version of the zebrafish genome and transcriptome
Supplementary_files_format_and_content: The processesd files are in Excel sheet format. The differential expression file include differential expression values (Log2Fold change) along with statistical significance. The rMATs file contain alternative splicing events analysis across wild type and fam50a KO mutants
|
| |
|
| Submission date |
Feb 21, 2020 |
| Last update date |
May 02, 2020 |
| Contact name |
Erica Davis |
| E-mail(s) |
[email protected]
|
| Organization name |
Ann & Robert H Lurie Children's Hospital of Chicago
|
| Department |
Advanced Center for Translational and Genetic Medicine (ACT-GeM)
|
| Street address |
225 E Chicago Avenue
|
| City |
Chicago |
| State/province |
IL |
| ZIP/Postal code |
60611 |
| Country |
USA |
| |
|
| Platform ID |
GPL21741 |
| Series (1) |
| GSE145711 |
Mutations in FAM50A suggest that Armfield XLID syndrome is a spliceosomopathy (zebrafish) |
|
| Relations |
| BioSample |
SAMN14162099 |
| SRA |
SRX7777136 |
