Methods and uses of high-throughput inference of synaptic connectivity relationships among cell types

A method of constructing a hyper-diverse barcoded plasmid library, comprising:

• a) amplifying a template plasmid in an amplification reaction using a forward primer and a reverse primer, wherein the forward primer and the reverse primer each comprise from 5′ to 3′ a complementary region, a barcode, a linker, and a restriction enzyme site, wherein the restriction enzyme sites of the forward primer and the reverse primer generate 3′ compatible overhangs when cleaved;
• b) generating double-stranded linear amplicons, each comprising the template plasmid sequence comprising barcodes at the termini of the amplicons;
• c) digesting the amplicon with a restriction enzyme to produce a digested product comprising 5′ and 3′ overhangs;
• d) ligating the digested product to produce a circular barcoded plasmid;
• and
• e) selectively digesting linear DNA with an exonuclease.