We used RNA sequencing (RNAseq) to identify potentially new expressed transcripts of ZNF804A and to guide the construction of relevant primers for 5′ rapid amplification of complementary DNA (cDNA) ends (5′ RACE). We performed RNAseq on Clontech pooled poly(A)+ mRNA from fetal and adult human brain to identify potential 5′ cDNA ends. After fragmentation, reverse transcriptase and random primers copied the cleaved fragments into first-strand cDNA. Second-strand cDNA was synthesized using T4 DNA polymerase, T4 polynucleotide kinase, and Klenow DNA polymerase, adding a single adenine base using a 3′ to 5′ exo− Klenow fragment, and ligating the paired-end adapters using T4 DNA ligase. An index (up to 12 nucleotides) was inserted into Illumina adapters so multiple samples could be sequenced in 1 lane of an 8-lane flow cell. Products were purified and enriched with polymerase chain reaction (PCR) to create the final cDNA library for highthroughput sequencing using the HiSeq 2000 system (Illumina). Results were mapped to GRCh37/hg19 using the TopHat2 splice-aware alignment software.31,32
