Transcriptome analysis of the immune system from Branchiostoma japonicum
Total cellular RNA was extracted with Trizol (Invitrogen). mRNA from 20μg RNAs was purified by binding to Oligo (dT) magnetic adsorption beads and fragmented into small pieces using divalent Ca ions at 94°C for 8 min. With these mRNA fragments as templates, the first-strand cDNA fragment was synthesized using random primers. The second-strand cDNA was produced using DNA polymerase I and RNaseH. Following end repair, adding poly (A) and ligation of sequencing adaptors, short cDNA fragments were purified with approximate 100 bp length and then further enriched with PCR to construct the final sequencing cDNA library