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Sample GSM8163706

Query DataSets for GSM8163706

Status

Public on Jul 29, 2024

Title

myd88_3

Sample type

SRA

Source name

endocardial

Organism

Danio rerio

Characteristics

cell type: endocardial
genotype: myd88-/-
time: 96 hpci

Extracted molecule

total RNA

Extraction protocol

RNA was isolated from FACS-sorted endocardial cells from a pool of 4 ET(krt4:EGFP); myd88+/+ and 4 ET(krt4:EGFP); myd88-/- ventricles for each sample using the miRNeasy micro Kit (Qiagen) combined with on-column DNase digestion (RNase-free DNase set, Qiagen) to avoid contamination by genomic DNA.
Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers, followed by the second strand cDNA synthesis using either dUTP for directional library or dTTP for non-directional library. For the non-directional library, it was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification. For the directional library, it was ready after end repair, A-tailing, adapter ligation, size selection, USER enzyme digestion, amplification, and purification. The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection. Quantified libraries will be pooled and sequenced on Illumina platforms, according to effective library concentration and data amount.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Data processing

Trimmomatic version 0.39 was employed to trim reads after a quality drop below a mean of Q15 in a window of 5 nucleotides and keeping only filtered reads longer than 15 nucleotides (Bolger et al., Trimmomatic: a flexible trimmer for Illumina sequence data).
Reads were aligned versus Ensembl zebrafish genome version danRer11 (Ensembl release 109) with STAR 2.7.10a (Dobin et al., STAR: ultrafast universal RNA-seq aligner).
Alignments were filtered to remove: duplicates with Picard 3.0.0 (Picard: A set of tools (in Java) for working with next generation sequencing data in the BAM format), multi-mapping, ribosomal, or mitochondrial reads.
Gene counts were established with featureCounts 2.0.4 by aggregating reads overlapping exons excluding those overlapping multiple genes (Liao et al., featureCounts: an efficient general purpose program for assigning sequence reads to genomic features).
The raw count matrix was normalized with DESeq2 version 1.36.0 (Love et al., Moderated estimation of fold change and dispersion for RNA-Seq data with DESeq2).
Assembly: danRer11
Supplementary files format and content: The tab-delimited matrix shows DESeq normalized gene counts.

Submission date

Mar 25, 2024

Last update date

Jul 29, 2024

Contact name

Noah Knoppik

Organization name

Max-Planck-Institute for Heart and Lung Research

Department

Bioinformatics

Street address

Ludwigstrasse 43