We used the RNAseq read alignment data to design primer pairs to amplify ZNF804A transcripts. We first performed 5′ RACE in the commercial fetal and adult brain poly(A)+ mRNA samples to validate the RNAseq results, with ZNF804A-specific antisense primers binding at exon 4, using SMART RACE cDNA Amplification and Advantage 2 PCR kits (Clontech). The poly(A)+ mRNAs were reverse transcribed according to the manufacturer’s protocol. The PCR amplification profile was as follows: 94°C for 2 minutes; 30 cycles of 94°C for 30 seconds, 70°C for 30 seconds, and 72°C for 2 minutes; and 72°C for 10 minutes after the last cycle. When necessary, nested PCR was done using gene-specific primers and a nested universal primer A. Having confirmed the 5′ cDNA ends, we used end-to-end PCR to validate the transcripts in the 2 samples by Advantage 2 PCR kits (Clontech) following the manufacturer’s recommendations. All products were cloned into pCR4-TOPO (Invitrogen) and sequenced.
