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Sample GSM8285552

Query DataSets for GSM8285552

Status

Public on May 22, 2024

Title

B_2_WT_ML233_3h_2

Sample type

SRA

Source name

embryo

Organism

Danio rerio

Characteristics

tissue: embryo
genotype: WT
developmental stage: 3 dpf embryo
time: 3h
mdibl id: B_2_WT_ML233_3h_2
conc um: 0.5
sequence count: 100003496

Growth protocol

Zebrafish were aplnr(a/b) mutants derived from the AB strain, and wildtype controls derived from the same grandparents. Husbandry and procedures were as described previously. All animal procedures were approved by the Institutional Animal Care and Use Committee of the MDI Biological Laboratory, and all methods were performed in accordance with the relevant guidelines and regulations. To generate tyrosinase, apelin, apelin receptor a, and apelin receptor b mutants, we used tracrRNA and crRNA (IDT) to form functional gRNA duplexes targeting the ORF (apln: 5’-GAATGTGAAGATCTTGACGC-3’, aplnra: 5’-TGGGTGTGACTACTCGGAGT-3’ and aplnrb: 5’- CTTGCAGAGTGCCACGCCAA-3’). Co-injection of these gRNAs with the Cas-9 protein (IDT) resulted in indels in the apln, aplnra, or aplnrb gene coding sequences, identified by Sanger sequencing and analyzed with ICE software (Synthego) in F0 injected embryos. F0 carriers for mutations in the apelin locus in the germline were identified by sequencing the alleles on the clutches, and were then outcrossed to obtain F1 heterozygous mutants. F2 apelin homozygous mutants were also identified by sequencing. The apelin mutant harbor a 26-bp insertion leading to a stop codon at position 18 of the peptide.

Extracted molecule

total RNA

Extraction protocol

Embryos were homogenized in Trizol solution (Invitrogen) and total RNA was purified using chloroform (Fischer) and then isopropanol (Fischer). RNA (1 μg) was reverse-transcribed using an oligo-dT primer and RevertAid kit (Thermo-Scientific). Total RNA samples for bulkRNAseq analysis were prepared with ~50 embryos/sample. Each experimental condition has been performed using biological quadruplicates to ensure reproducibility. Trizol (Invitrogen) was used to homogenize cellular extracts with the TissueLyzer (Qiagen). RNA purification was performed as described above.
RNA was quantified and sent to Novogene for Illumina library preparation and paired-end sequencing.
Messenger RNA was purified from total RNA using poly-T oligo-attachedmagnetic beads. After fragmentation, the first strand cDNA was synthesizedusing random hexamer primers, followed by the second strand cDNAsynthesis using dUTP.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Data processing

The library was checked with Qubit and real-time PCR for quantificationand bioanalyzer for size distribution detection.
Raw data (raw reads) in fastq format was firstly processed through in-house perl scripts. In this step, clean data (clean reads) was obtained by removing reads containing adapter, reads containing ploy-N and lowqualityreads from raw data. At the same time, Q20, Q30 and GC content were calculated
Paired-end clean reads were aligned to the reference genome GRCz11 (Danio rerio) using Hisat2 v2.0.5 < hisat2 -x reference_genome -p 4 --dta -t --phred33 -1 sample_1.clean.fq.gz -2 sample_2.clean.fq.gz --un-conc-gz sample.unmap.fq.gz 2> sample_align.log >
Mapped reads of each sample were assembled by StringTie [3](v1.3.3b) in a reference-based approach < stringtie sample.bam -p 4 -G genome.gtf -o sample.gtf --rf >
FeatureCounts [4] v1.5.0-p3 was used to count the reads numbers mapped to each gene. < featureCounts -T 4 -F GTF -t exon -g gene_id -s 2 -Q 10 -C -B -p -a genome.gtf -o feature_count.xls sample.bam >
Differential Expression Analysis was performed with the MDIBL Bioinformatics Core’s DESeq2 R script R version 4.3.2 and DESeq2 version 1.42.1. (parameters are contained in the report file: ML233_Simple_Base_Pairwise.html )
PCA Clustering was resolved using the R package PCAtools version 2.14.0
Gene Set Enrichment Analysis (GSEA) was performed using clusterProfiler version 4.10.1
Assembly: GRCz11 (Danio rerio) reference genome obtained from Ensembl
Supplementary files format and content: gene counts

Submission date

May 21, 2024

Last update date

May 22, 2024

Contact name

Thomas Dexter Morse

E-mail(s)

morse.t@northeastern.edu

Phone

2072338465

Organization name

MDIBL