IgSeq Sparus aurata: Naive4 anterior intestine
RNA from anterior intestine was extracted using the MagMax for microarray kit (Applied Biosystems) and reverse transcribed. Four representative fish per group (naive and immunized) were individually amplified using combinations of forward primers specific for each subgroup of IGHV genes and reverse primers specific for IGHM or IGHT genes. A library was constructed per individual with the TruSeq DNA PCR-Free Library Prep Kit (Illumina). Libraries were pooled together and paired-end sequencing was performed on an Illumina MiSeq with a MiSeq Reagent Kit v3 (2 x 300 cycles) cartridge (Illumina).