RNA sequencing

Embryos were treated at 13 hpf (8 somites) with TP-0903 and DMSO for 1, 4 and 8 h at 28°C. 35 embryos were collected for each treatment. 4 biological replicates were sequenced for each condition. The average read count per sample prior to mapping was 20 million. 92% (s.d. 0.04) of reads aligned to the reference; 68% (s.d. 0.08) aligned to a single location in the genome. Of the uniquely aligned reads, 75% (s.d. 0.02) aligned to known gene annotations. The average Pearson correlation between replicate samples was 0.9982. RNA was harvested using Qiagen RNeasy kit (cat. # 74104). Quality of RNA was assessed using Bioanalyzer RNA 6000 Nano Chip and RNA-Seq libraries prepared using Illumina TruSeq Stranded Total RNA Sample Prep Kit with Ribo-Zero (cat. # RS-122-2401). Sequencing was performed on an Illumina HiSeq 2000 using a 50-cycle single-read sequencing v3 kit (cat. # FC-401-3002).

USeq's MakeTranscriptome (v8.8.1) application was used to create all possible splice-junction sequences for each gene, using ensemble transcript annotations. The splice junction sequences were combined with zv9 chromosome sequences and used to create a Novoalign (v2.08.01) index. Reads were aligned to the reference using Novoalign, allowing up to 50 alignments for each read. The resulting alignment file was processed with USeq's SamTranscriptomeParser application, which selects the appropriate alignment location for each read and discards repetitive alignments or alignments with low qualities. SamTranscriptomeParser also converts splice-junction alignment coordinates back to genomic space. The processed alignments were then run through USeq's DefinedRegionDifferentialSeq (DRDS) application, which counts the number of alignments to each gene. Subsequently, differential expression analysis was generated on the count data with DESeq2 using default settings and a negative binomial distribution test (Love et al., 2014). All high-throughput sequencing data sets have been submitted to Gene Expression Omnibus (GEO) and can be accessed through GEO accession number {"type":"entrez-geo","attrs":{"text":"GSE72322","term_id":"72322"}}GSE72322 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE72322).