7ea782dd-9eab-459d-a4c5-f6fd45b36102
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 human brain and tissues. We performed a targeted GPHN RNA analysis using Third Generation Sequencing technology developed by Oxford Nanopore (ONT). RNA was extracted from 21 human tissues as described (Chomczynski P. et al., PMID: 17406285 ), and Reverse Transcription (RT) was performed with the specific primer GPHN-48: CAGGATACAGTCAATGATATGTGGACATGC. GPHN expression was next amplified using two rounds of PCRs, first 20 cycles, and after purification of PCR fragments a second PCR of 23 cycles was applied to each sample to add a barcode and allow multiplexing of the library. The multiplexed library was constructed using the Oxford Nanopore SQK-LSK109 kit and sequenced on MinION device.
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 human brain and tissues. We performed a targeted GPHN RNA analysis using Third Generation Sequencing technology developed by Oxford Nanopore (ONT). RNA was extracted from 21 human tissues as described (Chomczynski P. et al., PMID: 17406285 ), and Reverse Transcription (RT) was performed with the specific primer GPHN-48: CAGGATACAGTCAATGATATGTGGACATGC. GPHN expression was next amplified using two rounds of PCRs, first 20 cycles, and after purification of PCR fragments a second PCR of 23 cycles was applied to each sample to add a barcode and allow multiplexing of the library. The multiplexed library was constructed using the Oxford Nanopore SQK-LSK109 kit and sequenced on MinION device.