Fig. 3: Establishment of PBHL-seq for ROI expression analysis.

A The PBHL-seq workflow for in situ transcriptomic sequencing: (1) The sample is fixed and permeabilized. (2) In-situ RT is performed on fixed samples with primers containing NPOM-caged hybridization arm, unique molecular identifier (UMI), and dT oligo. (3–6) Within each ROI, a unique DNA barcode adaptor with T7 promoter, PCR handle, barcode N, and hybridization arm is hybridized and ligated to uncage-RT primers after UV illumination. (7) Double-strand is synthesized on the sample section, afterward, the double-strand cDNA is collected in an EP tube. (8) IVT, RT, and PCR amplification are performed. The sequencing library is prepared. NPOM eight 6-nitropiperonyloxylmethyl, ODNs oligodeoxynucleotides, RT reverse transcription, ROI region of interest, T7 T7 promoter, T7 Pol T7 polymerase, UMI unique molecular identifier, IVT in vitro transcription, RT-PCR reverse transcription-polymerase chain reaction. B Efficiency of hybridization by different hybridization buffers with multiplex analysis for ROIs. The ligation buffer contains 2×SSC, 10% formamide, 10% dextran sulfate sodium salt, Recombinant RNase Inhibitor, and primer. The tris buffer contains 0.3 M NaCl, 20 mM Tris-HCl pH 7.4, and 0.2% Tween 20. SSC buffer: Saline Sodium Citrate buffer. C, D Background from nonirradiated cells is performed with human-mouse mixed cultures. C DAPI staining marks the nuclei position of 293T and 3T3 cells (blue). Scale bar, 1000 μm. E Sequentially fluorescent labeling of multi-ROIs. Scale bar, 50 μm. F, G, H Cell mixing experiments with human 293T and mouse 3T3 cells show little species mixing. G After the second strand synthesis, the sample is stained by DRAQ5 (blue camouflage color). A subset of ~60 3T3 cells and ~80 293T cells are barcoded in the whole well (n  =  3 biological replicate, representative image shown). Scale bar, 250 μm. I Illustration of the experiments for DMD-assisted UV irradiation of 1, 10, ~100, and ~1000 cells. Scale bar, 250 μm. J, K Boxplot of numbers of gene-assigned UMIs and detected genes. Midline marks the median and edges indicate the 25th and 75th percentiles, and the meter shape represents the average value.