Computational protocol: Identification of the zebrafish maternal and paternal transcriptomes

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Protocol publication

[…] Embryos were collected 30 minutes after natural matings and then stage matched at the 2-cell, 64-cell, high (3.5 hpf), shield (6 hpf) and 90% epiboly (9 hpf) stages (). One hundred embryos were collected for each stage and total RNA was extracted using TRIzol (Invitrogen) and then DNase treated. The quality of total RNA was assessed using an Agilent Bioanalyser and quantified using a Qubit (Invitrogen). Polyadenylated mRNA was then extracted from total RNA and fragmented. A 200-300 nucleotide size selection was performed and the RNA was then converted into an Illumina sequencing library according to the manufacturer’s protocol (TruSeq, Illumina). Libraries were sequenced on a Genome Analyser II as 54 bp paired-end reads. TopHat and Cufflinks were used to analyse the sequencing results ().For the cycloheximide experiment, embryos were synchronised by in vitro fertilisation. Sperm samples were collected and stored on ice in 70 μl BSMIS (75 mM NaCl, 70 mM KCl, 2 mM CaCl2, 1 mM MgSO4, 20 mM Tris pH 8.0). Eggs were then collected into a glass Petri dish by squeezing females. Sperm samples were added to the eggs with the addition of 1 ml egg water. After 2 minutes the dish was flooded with egg water. Fertilised eggs were then split into two dishes, with cycloheximide being added to one dish at a final concentration of 200 μg/ml; cell cycle progression was blocked at this concentration. Untreated and treated embryos were then collected every 15 minutes, starting just after the addition of cycloheximide. RNA-seq was performed as described above. Throughout the experiment, care was taken to incubate both dishes at 28.5°C because cell cycle progression is affected by temperature. [...] Exome sequence variants were identified using a modified version of the 1000 Genomes Project variant calling pipeline (; ). Sequencing reads were mapped to the zebrafish genome using Burrows-Wheeler aligner (BWA) and SNPs were called by SAMtools mpileup, QCALL and the GATK Unified Genotyper. SNPs were removed if not called by all three callers and where the genotype quality was lower than 100 for GATK and lower than 50 for QCALL and SAMtools mpileup. The exome sequences of the male and female in each cross were compared and only homozygous differences were used in the identification of maternal and paternal mRNAs. Using those positions and our SNP calling pipeline, we counted the number of maternal or paternal SNPs in the RNA-seq experiments. Only positions that were informative in both crosses were used to determine whether a gene was maternally or paternally expressed. Within the RNA-seq SNP count, a count of two was used as a cut-off for determining whether an mRNA was maternal or paternal. […]

Pipeline specifications

Software tools TopHat, Cufflinks, Variant Calling Pipeline, BWA, SAMtools, QCALL, GATK
Application RNA-seq analysis
Organisms Danio rerio