Cre-dependent massively parallel reporter assay for cell-type specific assessment of the functional effects of 3'UTR genetic variants in vivo

Here, we examined de novo variants identified within annotated 3'UTRs from the whole-genome sequencing of 519 families from the Simons Simplex Collection (Werling et al., 2018), totaling 342 mutations from probands and 299 from unaffected siblings within the same cohort. For each variant we synthesized an allelic pair of 3'UTR 'elements' spanning 120 bp of sequence centered on the variant. To be able to compare biological to non-biological sequences, for 322 variants, we randomly shuffled the sequence to generate a set of GC-matched controls. We tagged all 1,624 elements with six unique barcodes to provide internal replicates and control for potential barcode effects. To enable eventual cell-type specific studies, we cloned the final library of 9,744 synthesized oligos into the 3'UTR of a membrane-localized tdTomato reporter embedded in a Double-floxed inverse Orientation (DiO) cassette (Schnütgen et al., 2003), such that the reporter library would only express following Cre-mediated recombination. We completed the analysis of this library in vitro in N2a cells and in vivo in Vglut-Cre mice. Additional code and results corresponding to this GEO submission can be found at https://bitbucket.org/jdlabteam/asd_3utr_mpra_analysis/src/master/ Overall design: 6 replicates of N2a cells, 11 replicates of Vglut-cre mice,6 replicates of Vgat-cre