Dataset features


Application: Flow cytometry
Number of samples: 20
Release date: Oct 2 2018
Last update date: Nov 28 2018
Access: Public
Taxon: Homo sapiens
Dataset link A multiplexed assay for exon recognition reveals that an unappreciated fraction of rare genetic variants cause large-effect disruptions to splicing

Experimental Protocol

MFASS is a splicing minigene reporter. There are three synthetic oligo libraries, SRE library in two backbones and the SNV library. The oligo library is cloned in between a split GFP reporter with a downstream mCherry reporter. Exon inclusion leads to both GFP and mCherry fluorescence, exon skipping leads to only mCherry fluorescence. We sort the libray of cells based on these two fluorescent reporters using fluorescence activated cell sorting (FACS). We then sequence each individual bin to get a quantitiative measure of splicing. For the SRE library, we sort into three bins, with two replicates. Double positive (DP) indicates both GFP and mCherry are present and correspond to exon skipping. Intermediate (INT) indicates intermediate levels of GFP and mCherry. Single positive (SP) indicates no GFP present but mCherry present, corresponds to exon inclusion. For the SNV library, the sorting is similar except there are four bins, also done in replicate. Hi indicates both GFP and mCherry, Int-Hi and Int-Lo correspond to two intermediate bins, and Lo corresponds to no GFP present but mCherry present. We use the read counts across all bins, present in processed data files, to calculate an inclusion index for an individual sequence.










Kimberly Insigne