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Sample GSM3581496

Query DataSets for GSM3581496

Status

Public on Jan 29, 2019

Title

Harlequin duck Replicate 3

Sample type

SRA

Source name

Trigeminal Ganglion

Organism

Histrionicus histrionicus

Characteristics

age: Embryonic Day 28-29

Growth protocol

Eggs of bird species were incubated at 37°C and 55%-75% humidity. Embryos were extracted for dissection when they had broken through the inner shell membrane (24-48 hrs before hatching)

Extracted molecule

total RNA

Extraction protocol

Trigeminal ganglia were dissected from embryos, flash-frozen and stored at -80°C until RNA isolation. Total RNA was isolated from the stored trigeminal ganglia using the TRIzol reagent (ThermoFisher, Waltham, MA) according to manufacturer’s instructions. RNA quality and integrity was confirmed by running on Agilent Bioanalyzer, with RINs in the range of 7.7-8.6.
mRNA was purified from ~500 ng total RNA with oligo-dT beads. Strand-specific sequencing libraries were prepared using the KAPA mRNA Hyper Prep kit (Roche Sequencing, Pleasanton, CA).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Description

Harlequin duck

Data processing

Primary analysis performed using Illumina's CASAVA 1.8.2 software suite.
Raw sequencing reads were filtered and trimmed to retain high-quality reads using Trimmomatic v0.36 (Bolger et al., 2014) with default parameters
Filtered high-quality reads from all samples were aligned to both duck and chicken reference genomes using STAR aligner v2.5.4b with default parameters (Dobin et al., 2013).
Aligned reads were counted by featureCounts program within the Subread package v1.6.2 with default parameters (Liao et al., 2014).
Raw read counts were processed and converted to “reads per kilobase gene length per million mapped reads” (RPKM) values by EdgeR v3.22.3 (Robinson et al., 2010).
To compare gene expression estimates between samples from different species, gene lists and corresponding RPKM values from duck and chicken gene annotations were merged based on the common gene symbol.
Supplementary_files_format_and_content: Raw read counts were processed and converted to “reads per kilobase gene length per million mapped reads” (RPKM) values by EdgeR v3.22.3. RPKM values from 3 biological replicates within each species were averaged and reported in the final data table. To compare gene expression estimates between samples from different species, gene lists and corresponding RPKM values from duck and chicken gene annotations were merged based on the common gene symbol. To maximize the number of matching gene symbols between annotations from different species, gene symbols from the NCBI annotation were converted to consensus gene symbols using the following gene ID conversion databases: DAVID Bioinformatics Resources 6.8 (https://david.ncifcrf.gov/home.jsp), Ensembl databases of gene orthologs (www.ensembl.org), and the NCBI gene symbol synonyms database (ftp://ftp.ncbi.nlm.nih.gov/gene/DATA/gene_info.gz). In case of gene orthologs with a common gene symbol between duck and chicken annotations, final count data for samples from all duck species comes from reads aligned to the duck genome, while count data for samples from chicken comes from reads aligned to the chicken genome. In case of genes without a matching gene ortholog in the other annotation (i.e. for duck genes without an ortholog in chicken, and vice versa), final count data for all samples comes from reads aligned to the corresponding genome only (i.e. only to duck genome in case of duck genes). The final data table (in .xlsx format) contains averaged RPKM values for each sample for each gene in the merged gene annotation as described above. Description of final data table columns: GeneSymbol_Consensus: a common gene symbol after merging duck and chicken annotations; GeneSymbol_Duck: original gene symbol from the annotation (.gff file) for duck genes; NCBI_Gene_ID_Duck: corresponding Gene ID from NCBI Gene Database; GeneSymbol_Chick: original gene symbol from the annotation for chicken genes; NCBI_Gene_ID_Chick: corresponding Gene ID from NCBI Gene Database; SourceGenome: source of data values for a particluar gene as described in the merging strategy (i.e. from reads aligned to the Duck or Chicken genome, or both, depending on whether the common gene symbol was present in the annotations). S1-S7_rpkms (Species): average RPKM values over 3 replicate samples for the given species.

Submission date

Jan 28, 2019

Last update date

Feb 04, 2019

Contact name

Viktor Feketa

E-mail(s)

viktor.feketa@yale.edu

Organization name

Yale University

Department

Cellular & Molecular Physiology