Figure 1. Transgenic variant expression and in situ sequencing with VIS-seq.

(A) VIS-seq uses fluorescent in situ sequencing of abundant circular RNA barcodes to genotype cells expressing protein variants. (1) A variant library in the VIS-seq expression cassette is integrated into cells via piggyBac-ase. (2) Cells are fixed; barcodes are reverse transcribed, captured with a padlock probe and amplified; (3) cells are stained and imaged; (4) barcodes are sequenced in situ; (5) single cell phenotype-genotype pairs are determined using STARCall; and (6) features for each cell are extracted using CellProfiler.

(B) VIS-seq expression cassette (PB = piggyBac³⁴; PD = padlock probe¹³⁰ binding site; UCOE = universal chromatin opening element²⁸; HBB IVS2 = hemoglobin subunit beta intervening sequence 2^29,30; IRES = internal ribosome entry site¹³¹; WPRE = woodchuck hepatitis virus post-transcriptional regulatory element³¹; cHS4 = chicken beta globin locus control region hypersensitive site 4²⁷).

(C) Time-series plot of mEGFP positivity of human WTC11 iPS cells and derived cardiomyocytes. mEGFP-tagged WT histone H1.4 is expressed via the VIS-seq cassette (blue) or lentivirus (red). Two replicates are plotted.

(D) Violin plot of the number of rolling circle amplification colonies per cell after one cycle of sequencing for VIS-seq expression cassette (blue) and lentivirus (red) cells from (C) at day 14. Silenced cells were excluded. Two replicates are shown.

(E) Matched images of mEGFP-tagged lamin A variants in U2OS cells, stained with DAPI, phalloidin, WGA, and mitoprobe (left), with corresponding first base in situ sequencing; magenta=guanine, blue=thymidine, yellow=adenine, and red=cytosine (right). Silver dashed lines depict the borders of cells. Scale bar indicates 10 μm.

(F) Matched images of mEGFP-tagged PTEN library in human WTC11 PTEN-KO inducible-NGN2 iPS cells, stained with DAPI and phalloidin-CF568 (left, top row) and first base in situ sequencing (right, top row). Matched images of mEGFP-tagged PTEN library in neuron-like cells, seven days after induction of NGN2, stained with DAPI and anti-MAP2 AF568 (left, bottom row) and first base in situ sequencing (right, bottom row). Silver dashed lines depict the borders of iPS cells (top) or cell bodies of NGN2-induced neuron-like cells (bottom). Nucleobase coloring identical to (E). Scale bar indicates 10 μm.