Targeted long read sequencing of neuronal cell surface receptor encoding genes
C57Bl6/J mice were anesthetized with isoflurane or cryoanesthesia (neonates only) followed by decapitation. Eyes were enucleated and retinas were dissected out, or brain was dissected from the skull and the cerebral cortex was removed. Total RNA was isolated using Tri Reagent (ThermoFisher Scientific AM9738) according to the manufacturers protocol. Tissue was mechanically homogenized in Tri Reagent followed by phase separation with chloroform and isopropanol precipitation. RNA samples were stored at -80OC. RIN number was calculated using a Bioanalyzer. Only RIN values above 9 were used for sequencing. Reverse transcription was carried out using the Clontech SMARTer cDNA kit according to the manufacturers protocol. cDNA was amplified with KAPA HiFi DNA Polymerase for 12 cycles followed by size selection (4.5 to 10 Kb). For capture, 1 ug of cDNA was denatured and blocked with DTT primer and Clontech primer then mixed with Nimblegens SeqCap EZ Developer (200Mb) custom baits at 47OC for 20 hrs. Biotynaylated cDNAs were pulled down with streptavidin beads and washed with Nimblegen hybridization buffers to minimize non-specific binding. Targeted cDNA library was amplified 11 cycles with Takara LA Taq. SMRT bell library was constructed then additional size selection was performed (4.5 to 10Kb) followed by binding of Polymerase with P6-C4 chemistry (RSII). Library was loaded onto SMRT cell using MagBead loading at 80pM (RSII).