Extended Data Fig. 10: OligoFISSEQ applications.
From: 3D mapping and accelerated super-resolution imaging of the human genome using in situ sequencing
a, O-eLIT and immunofluorescence (IF). 36plex-1K was sequenced 5 rounds with O-eLIT off Mainstreet. Then, the same sample was prepared for IF and stained with antibodies. Samples were counterstained with wheat germ agglutinin (WGA) to stain membranes. Images are from deconvolved, maximum z-projections representative of 2 replicates. b, Chromosomal regions imaged with OligoSTORM from Fig. 6d enlarged and displayed separately. Orientation may differ from Fig. 6d. n = 1. c, 8 rounds of O-LIT sequencing of Chr19-9K off of Mainstreet. Images are maximum z-projections. Signal is detectable in all rounds even though the imaging was conducted without the advantage of eLIT, suggesting that 8 rounds of O-eLIT will produce even stronger signals. Images are representative of 2 replicates. d, O-LIT is compatible with gel embedding and target amplification via rolling circle amplification (RCA). Chr19-9K was hybridized to PGP1f cells, after which the sample was embedded in a hydrogel and then cleared of cellular background with proteinase. Next, a molecular inversion probe (MIP) was hybridized to a Chr19-9K specific barcode on Backstreet as well as a fluorophore labeled (purple) secondary oligo to Mainstreet to visualize Chr19-9K Oligopaint oligos. MIPs were circularized via ligation and RCA, after which the first digit of the barcode was sequenced using O-LIT (green). Images are representative of two replicates. e, Comparison of secondary fluorophore signal (2o) versus first round sequencing signal (LIT) from puncta in panel b images. Center values are mean values (3.4 for 2o and 4.9 for O-LIT) with SD.
