Fig. 1: NIS-Seq enables optical barcode identification in nucleated cells.

a, Outline of NIS-Seq reaction steps, adding a T7 transcription step to previously established in situ sequencing of barcoded mRNA¹⁰. b, First-cycle NIS-Seq imaging results in comparison to cytosolic in situ sequencing results obtained across eight cell types. Live cells were stained for nuclei (gray) and membrane (red) before fixation (left panels). Scale bar, 50 µm. Representative images from two experimental replicates are shown. c, Raw images of 14 cycles of NIS-Seq barcode sequencing from THP1 cells. Nuclear staining was performed at cycles 1, 4, 7, 10 and 13. Scale bar, 10 µm. Representative data from four experimental replicates are shown. d, Quantitative spot intensities obtained from THP1 cell nuclei I and II highlighted in c. Indicated on top is the base calling result, matching two members of the pooled lentiviral library used. e, Fraction of cells mapping to known library member sequences. Scrambled controls indicate analysis results using a permutated reference library with equal base distribution. Dots indicate analysis results from two halves of a 0.56-cm² surface. Two rows of edge tiles were excluded from analysis to avoid empty or distorted images. f, Specificity and sensitivity of NIS-Seq was quantified using a mixed population of cells either expressing GFP or containing NIS-Seq-compatible genomic insertions. The standard NIS-Seq protocol was compared to a modified version using PFA fixation and subsequent de-crosslinking (Methods). Dots indicate analysis results from two independent wells. g, Library coverage in transduced THP1 macrophages, measured by PCR-based NGS and NIS-Seq. Each library member covered in PCR-based sequencing is represented by one dot; dropping out sgRNAs—for example, those targeting essential genes—are not shown. Jitter was added to values to visualize spot density at low integer values.