Computational protocol: Transcriptome Profiling Identifies Ribosome Biogenesis as a Target of Alcohol Teratogenicity and Vulnerability during Early Embryogenesis

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Protocol publication

[…] Analysis of sequencing data was performed as described previously [], with minimal modification. In brief, RNA-Seq sequence reads were filtered through 4 trimming procedures that removed: (i) reads failing to meet or exceed Illumina’s default chastity threshold of 0.6 over the first 25 cycles; (ii) reads containing more than 2 contiguous or interspersed ambiguous nucleotides; (iii) reads with quality scores <20 (Phred scale; 1 error per 100 nucleotides); and (iv) reads that were shorter than the expected 75 bp. The RNA-Seq module in CLC Genomics Workbench 5.5 (CLC Bio, Cambridge, MD) was used to map the filtered paired-end reads to 17,108 genes (known or predicted) derived from assembly 4.0 of the Gallus gallus genome (Ensembl release 73; September 2013). Reads were mapped to the reference genome, individually requiring at least 90% of each read to exhibit 80% or greater alignment similarity. Only reads that mapped uniquely to the reference were included in subsequent analyses. Raw counts of read mappings were normalized to the reference genome using DESeq v1.10.1 [] as described previously [].All p-values were adjusted with the Benjamin-Hochberg multiple testing correction to control the false discovery rate (FDR). Data were evaluated as mean normalized counts vs. dispersion, and as mean normalized counts vs. log2 fold change, plotting both empirical dispersion and fitted values for the former. Because DESeq is generally considered to use a conservative algorithm, genes were deemed differentially expressed if the adjusted level of significance was below 0.10. Genes identified as differentially expressed (DE) were submitted to DAVID [,] for GO term analysis. Using the Kyoto Encyclopedia of Genes and Genomes KEGG [], pathway analysis was also performed in DAVID v6.7 using the above parameters.Our previous comparison [] of differential gene expression in the neural folds of experimentally-naïve alcohol-vulnerable (Hy-Line W98S) and alcohol-resistant (Hy-Line W98D) embryos used galgal3 release e70 (Ensembl, January 2013). Because gene annotation often improves with new database releases, we re-analyzed those data by mapping the same RNA-Seq reads to galgal4 release e73 (Ensembl, September 2013) as above, such that all data sets would be consistent with the revised genome assembly in the galgal4 release. We then determined the sets of differentially expressed genes in both datasets. An enrichment analysis for both GO terms and KEGG pathways was then performed on this set of differentially expressed genes as described above. Finally, we determined the intersection of differentially expressed genes in the vulnerable/responder sets and tested those genes for GO term and KEGG pathway enrichment. All data utilized herein from study [] thus represent a reanalysis using the updated galgal4 release e73. [...] Translation-blocking morpholinos were designed and purchased from Gene Tools (Philomath, OR). Sequences used in this study were (5’-TTTGCCGACTGCCATGTGAACAC-3’) for RPS3A (MO1-rps3a) [], (5’-ACCCATTTTGTGATCGTTTGTTCTC-3’) for RPL5A (MO1-rpl5a) [], and (5’-CTTCTTCTCGCTCTGGTCCGCCATG-3’) for RPL11 (MO1-rpl11) [,]. A standardized nonsense morpholino (5’-CCTCTTACCTCAGTTACAATTTATA-3’) was used as an irrelevant sequence control. Zebrafish (Danio rerio) eggs (outbred strain 5D, gift of R. Tanguay, Oregon State University) at the 1-cell or 2-cell stage were collected and sorted from an on-site population of breeders. Zebrafish studies were reviewed and approved by the UW-Madison IACUC under protocol #A005104. Approximately 5 nl of morpholino diluted in 1X Danieau solution was injected into the yolk with a beveled 20 μM inner-diameter bore micropipette (Origio A/S, Denmark) that was connected to an ASI MPPI-3 pulse injector (Eugene, OR). The concentration of each morpholino injected was 30 μM (rps3a), 600 μM (rpl5a), 0.0625 μM (rpl11), and 100 μM (control), and were defined empirically such that they did not cause appreciable apoptosis or facial dysmorphology, minimizing risks for off-target effects []. At 70% epiboly, chorionated embryos were exposed for 3 hrs to 241 mM ethanol (1.5%) in fish water, and this dose produces an alcohol concentration within the embryo of 86 mM (0.5%) []. This dosage and exposure window causes a calcium-dependent neural crest apoptosis and facial malformations in zebrafish embryos which parallel those of the chick model []. At four days post-fertilization (dpf), embryos injected with either rps3a or rpl5a were euthanized and stained with Alcian Blue following the protocol described in Carvan, et al., []. Injection into the embryo of rpl11 morpholino at concentrations at greater than 0.25 μM were lethal or caused abnormal development at 24 hpf. When the concentration of rpl11 morpholino was reduced to 0.0625 μM, embryos survived and were morphologically comparable with control embryos through 3 dpf, at which time they were processed in the same manner as RPS3A and RPL5A.The DeadEnd Fluorometric TUNEL System (Promega, WI) was used to visualize fragmented DNA in apoptotic cells. Zebrafish embryos were injected with rps3a, rpl5a, or rpl11 morpholino as described above. Embryos were enzymatically dechorionated with Pronase [] at 4hpf, and treated at 75% epiboly with 42 mM alcohol for three hours. The alcohol was then washed away and the embryos were incubated an additional 2hr without alcohol and then were fixed at 12hpf (~4–6 somites) and stained for TUNEL at 12hpf as previously described []. Enumeration of TUNEL+ cells in 10–12 embryos per treatment was analyzed using a one-way analysis of variance followed by pairwise multiple comparisons using the Holm-Sidak method (SigmaPlot, San Jose CA). […]

Pipeline specifications

Software tools CLC Genomics Workbench, DESeq, DAVID, SigmaPlot
Databases KEGG KEGG PATHWAY
Applications Miscellaneous, RNA-seq analysis
Organisms Danio rerio, Gallus gallus, Caenorhabditis elegans, Homo sapiens
Chemicals Ethanol