Alternative splicing generates a myriad of Gephyrin isoforms at iPSD

Gephyrin (GPHN) regulates the clustering of postsynaptic components at inhibitory synapses and is involved in pathophysiology of neuropsychiatric disorders. Here, we uncover an extensive diversity of GPHN transcripts that are tightly controlled by splicing during mouse brain development. We performed a targeted Gphn RNA analysis using Third Generation Sequencing technology developed by Pacific Bioscience (PACBIO). RNA was extracted from mouse cortex and cerebellum tissues as described (Chomczynski P. et al., PMID: 17406285 ), and Reverse Transcription (RT) was performed with the specific primer GPHN-49: GTACTGTGCCTGAGGCTGC. Gphn expression was next amplified using two rounds of PCRs, first 20 cycles with the primers GCAGTCGAACATGTAGCTGACTCAGGTCACCCACGACCATCAAATCCGTGTC and TGGATCACTTGTGCAAGCATCACATCGTAGCAGGATACAGTCAATGATATGTGGACATGC, and after purification of PCR fragment a second PCR of 23 cycles was applied to each sample to add a barcode and allow multiplexing of the library.