Transcriptome Assembly of Horseshoe Crab Embryos

The cDNA library was prepared using the TruSeq^TM RNA Sample Preparation Kit (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. Poly(A)-containing mRNA was purified by Oligo(dT) magnetic beads from 10 μg total RNA sample and fragmented using divalent cations. The cleaved RNA fragments were used for the first strand cDNA synthesis using reverse transcriptase and random primers, followed by second strand cDNA synthesis using DNA polymerase I and RNase H. After second strain cDNA synthesis, fragments were treated with end repair, A-base tailing and adapter ligation consecutively. The sample was further treated by gel size fractionation and PCR amplification to create final cDNA library. The cDNA library was sequenced on the Illumina Cluster Station and Illumina Genome Analyzer system according to the manufacturer’s instructions. The Trinity method was used for de novo assembly of Illumina reads of T. tridentatus embryos [27 (link)]. Briefly, the trinity using de Bruijn graph algorithm was run on the paired-end sequences with the fixed default k-mer size of 25, minimum contig length of 200 and paired fragment length of 500.