Pcr4 topo backbone

NRXN1α cDNA was generated as previously described for targeted short-read sequencing. Gel extracted NRXN1α cDNA was ligated into the pCR4-TOPO backbone (ThermoFisher) following the manufacturers guidelines. Individual colonies were picked and screened for full length NRXN1α isoforms by Sanger sequencing (Genewiz). Two specific NRXN1α isoforms in the pCR4-TOPO backbone were chosen for further cloning into a lentiviral expression vector. A 4.5-kb fragment containing most of coding sequence of NRXN1α was amplified from the pCR4-TOPO backbone. An upstream region of the first coding exon and a downstream region of the last coding exon fused to a 3xFLAG tag were amplified from two synthetic plasmids containing these sequences (Thermofisher). Fragment amplification was performed using Q5 polymerase following manufactures protocol using appropriate primers (Supplementary Table 19). Fragments were assembled using the NEBuilder HiFi DNA Assembly Master Mix following the manufacturers protocol and the completed assembly was transformed into Stbl3 Chemically Competent E. coli (ThermoFisher), and positive clones were confirmed by restriction digest and Sanger sequencing (Genewiz). Lentiviruses were produced from these vectors as previously desceribed²² . Physical titration of lentivirus was performed by using a qPCR Lentivirus Titration Kit (ABM).