4 protocols
data generation
Illumina standard protocol was used.
The hybrid F1 of Siniperca scherzeri (Siniperca chuatsi (body length about 5 cm per fish) were obtained from Guangdong Freshwater Fish Farm (Panyu, Guangdong Province, China). The training was performed by following the methods reported by Liang et al. (2001) using net-cages as the experimental culture. Fry of India mrigal Cirrhina mrigola were used as the live prey fish for mandarin fish in this study, and the dead prey fish were prepared by freezing. During the training period, the fish were visually sorted into feeders and nonfeeders on the basis of plumpness or emaciation, respectively. The training period did not cause the nonfeeders to die of hunger because of the relatively short length of the time and large size of the fish. To eliminate the influence of hunger on the transcriptional levels of mandarin fish, both two groups were fed with live prey fish for two days after training
Equal masses of total RNA extraction for brain and liver tissues of 16 individuals for dead prey fish feeders and nonfeeders in mandarin fish was performed using SV total RNA isolation system (Promega, USA) according to manufacturer's protocol. Libraries for brain and liver tissues were constructed according to the standard protocol by Beijing Genomics Institute (BGI). Beads with Oligo(dT) are used to isolate poly(A) mRNA after total RNA is collected from eukaryote (prokaryocyte can be treated with kit to remove rRNA before next step). Fragmentation buffer is added for interrupting mRNA to short fragments. Taking these short fragments as templates, random hexamer-primer is used to synthesize the first-strand cDNA. The second-strand cDNA is synthesized using buffer, dNTPs, RNaseH and DNA polymerase I, respectively. Short fragments are purified with QiaQuick PCR extraction kit and resolved with EB buffer for end reparation and adding poly(A). After that, the short fragments are connected with sequencing adapters. And, after the agarose gel electrophoresis, the suitable fragment are selected for the PCR amplification as templates. At last, the library could be sequenced using Illumina HiSeq 2000.
Illumina standard protocol was used.