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Sample GSM7505564

Query DataSets for GSM7505564

Status

Public on Aug 10, 2023

Title

WT_EF_set_1_S5

Sample type

SRA

Source name

liver

Organism

Danio rerio

Characteristics

tissue: liver
genotype: WT
time: 7dpf
treatment: overfed animal

Extracted molecule

total RNA

Extraction protocol

Total mRNAs were extracted from pools of dissected livers from 6 dpf abcb11b mutants and WT siblings, 20 livers per replicate and 3 replicates per genotype, by using Arcturus PicoPure RNA isolation kit (Applied Biosystems, KIT0204). For the 40 livers per replicate (4-5 replicates per group) were dissected under a brightfield microscope at 7dpf and then pooled prior to RNA extraction using the Qiagen RNAeasy Micro kit. Animals in the overfed group were provided with six times the normal amount of food at each feeding from day 5 to 7 while EtOH treated animals were exposed to 2% ethanol for 32 hours prior to sample collection. The presence of hepatic steatosis was confirmed in both groups.
WT and abcb11b mutants samples were submitted to Novogene Co for library preparation and RNA-sequencing using the vendor’s standard protocol. The 40 samples were submitted to the Centre for Applied Genomics (TCAG, Toronto, Canada). SMART-Seq v4 Ultra Low Input RNA kit was used for the cDNA conversion. Library preparation followed the protocol of Nextera XT library prep kit. Libraries were quality controlled (QC)ed (run on the Bioanalyzer DNA High Sensitivity chip to check for size and quantified by qPCR using Kapa Library Quantification Illumina/ABI Prism Kit protocol). All libraries were pooled and sequenced on 2 lanes on the NovaSeq SP flowcell paired-end 2x100bp.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

NextSeq 2000

Data processing

Fastq files from the different runs were merged using “cat”. The merged paired FASTQ files for each sample were run through FastQC (version 0.11.09) to obtain general QC metrics. Adapters were trimmed by Trim Galore (version 0.6.6). Salmon3 (version 1.4.0) was used for our alignment-free pipeline. Adapter-trimmed reads were used as input.
We quantified gene expression using raw counts and kept the genes that showed at least 5 reads in 80% of the samples.
We performed differential expression gene testing with DESeq2 (v.1.24.0 R package) using default settings. Replicate was used as covariates within the DESeq2 model. Statistical significance was set a 5% FDR (Benjamini-Hochberg).
The Bioconductor package fgsea (v 1.10.1 R package), was used for gene set enrichment analysis (GSEA).
Assembly: Danio Rerio reference genome (GRCz11) with associated transcript annotations (Gencode version GRCz11.103) was used.
Supplementary files format and content: Salmon quant files for liver samples from WT and abcb11b mutants
Supplementary files format and content: Raw counts for the 40 livers per replicate

Submission date

Jun 22, 2023

Last update date

Aug 10, 2023

Contact name

Kristen Seim

E-mail(s)

kristen.seim@gmail.com

Organization name

GraphiteBio

Street address

201 Haskins Way