Computational protocol: A conserved maternal-specific repressive domain in Zelda revealed by Cas9-mediated mutagenesis in Drosophila melanogaster

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Protocol publication

[…] RNA-seq experiments were done on hand-sorted stage 5 embryos laid by zldZnF2 (C554S mutation) homozygous females and w1118 embryos as a wild-type control. Embryos were dechorionated, analyzed under halocarbon oil to determine stage, collected and lysed in TRIzol (ThermoFisher) supplemented with 150 μg/ml glycogen. RNA was extracted, and cDNA libraries were prepared using Truseq RNA sample prep kit (Illumina). Three replicates of each were sequenced. The cDNA 100 bp single-end reads were sequenced at the UW Biotechnology Center DNA Sequencing Facility using an Illumina HiSeq 2000. Using the Galaxy platform [], reads were examined for quality, trimmed, and filtered. The reads were then mapped to the BDGP D. melanogaster (dm6) genome using RNA-STAR (Galaxy Version 2.5.2b-0). Cufflinks (Galaxy Tool version 2.2.1) was used with default settings for transcript assembly. The resulting assembled transcripts were compared using Cuffdiff [](R version 3.1.2) to identify genes that change significantly (p-value<0.05, >two-fold change) in expression. Only genes that were significantly mis-expressed in all replicates were used for further analysis.Prior to comparisons, all gene IDs were converted to current FlyBase identifiers (FBgn#) using the ‘Upload/Convert IDs’ tool available on FlyBase []. Single-embryo expression data from Lott et al. 2010 [] were used to classify up- or down-regulated genes in mutant embryos as (1) zygotic, (2) zygotic-maternal, and (3) maternal only. Translation and stability datasets of maternal mRNAs from Thomsen et al. 2010 [] were used to classify up-regulated maternal genes as targets of maternal degradation or zygotic degradation. mRNAs degraded by the maternal pathway were considered as Classes II and III (‘exclusively maternally degraded’ and ‘maternally degraded and transcribed’); and mRNAs degraded by the zygotic pathway, were considered as Class IV (‘exclusively zygotically degraded’). ZLD ChIP-seq data from Harrison et al. 2011 [] were used to identify the number of down-regulated zygotic genes bound by ZLD. Enrichments and depletions for comparisons to data from Lott et al. 2010 [], Thomsen et al. 2010 [], and Harrison et al. 2011 [] were determined using a Fisher’s exact test. […]

Pipeline specifications

Software tools Galaxy, STAR, Cufflinks
Databases BDGP
Application RNA-seq analysis
Organisms Drosophila melanogaster, Caenorhabditis elegans
Chemicals Zinc