Fig. 1: The PTRE-seq MPRA library undergoes pervasive cryptic splicing.

A Schematic of the PTRE-seq library. Original “barcode” primers and “extended” primers used in this work are shown at bottom. eGFP, enhanced green fluorescent protein. The position of the palindromic insertion is indicated. B Size heterogeneity was observed in sequencing libraries prepared from the PTRE-seq DNA plasmid (grey and orange, denoting two different PCR protocols) and RNA (blue). Electropherogram was measured by Tapestation 4200. C Representative read coverage tracts of reporters with internal deletions in full PTRE-seq library. D Position weight matrices of sequences observed at 5’ and 3’ deletion boundaries observed in PTRE-seq reporters and annotated 5’ and 3’ splice sites retrieved from the human GRCh38 genome assembly. E Quantification of read categories from sequencing of the PTRE-seq libraries prepared from RNA reverse-transcribed with SuperScript II (SSII). Values denote the mean percentage for each category over two biological replicates. F Distribution of observed splicing fractions of PTRE-seq reporters. G Pearson correlations of PTRE-seq observed splicing fractions measured across biological replicates of the same cell line (diagonal, green outline) and between different cell lines.