Macrobrachium rosenbergii developmental transcriptomes
Total RNA from 200 M. rosenbergii larvae (Zoea 4 to 11) and PL2-28 was isolated. Equal amounts of RNA were pooled into four pools, each containing RNA from 40-60 individuals of 5 larval or PL sub-stages (8-15 individuals at each sub-stage), in duplicate. To ensure representation of low abundance transcripts in the transcriptome assembly, each of the four pools was divided into two tubes of which one was subjected to enzymatic normalization. All eight samples were used for the de novo assembly, while differential expression was calculated only for the four non-normalized pools. These eight total samples were subjected to next generation sequencing by Fasteris Life Science (Geneva, Switzerland) on an Illumina Genome Analyzer (HighSeq 2000, Illumina, San Diego, CA), performing 100 bp–paired end sequencing on one lane. The sequence reads were separated into the 8 libraries according to their indexes (de-multiplexing) and stored as FASTQ files. This yielded a total of ~242 million reads, with 14-38 (average 30) million reads per library.  To create a reference transcriptome, all reads were de novo assembled using Velvet, with several K-mer values, followed by Oases. The pair-read insert average size was set at 200 bases, with a standard deviation of 10%.