Hybridization-based in situ sequencing (HybISS) for spatially resolved transcriptomics in human and mouse brain tissue

Abstract

Visualization of the transcriptome in situ has proven to be a valuable tool in exploring single-cell RNA-sequencing data, providing an additional spatial dimension to investigate multiplexed gene expression, cell types, disease architecture or even data driven discoveries. In situ sequencing (ISS) method based on padlock probes and rolling circle amplification has been used to spatially resolve gene transcripts in tissue sections of various origins. Here, we describe the next iteration of ISS, HybISS, hybridization-based in situ sequencing. Modifications in probe design allows for a new barcoding system via sequence-by-hybridization chemistry for improved spatial detection of RNA transcripts. Due to the amplification of probes, amplicons can be visualized with standard epifluorescence microscopes for high-throughput efficiency and the new sequencing chemistry removes limitations bound by sequence-by-ligation chemistry of ISS. HybISS design allows for increased flexibility and multiplexing, increased signal-to-noise, all without compromising throughput efficiency of imaging large fields of view. Moreover, the current protocol is demonstrated to work on human brain tissue samples, a source that has proven to be difficult to work with image-based spatial analysis techniques. Overall, HybISS technology works as a targeted amplification detection method for improved spatial transcriptomic visualization, and importantly, with an ease of implementation.

Figure 1.

HybISS method overview, hybridization-based in situ sequencing. (A) Overview of ISS, first reverse transcribing mRNA transcripts to cDNA. Gene specific PLPs target cDNA with juxtaposed ends next to each other that allow for ligation of PLP. Only transcripts that are ligated are enzymatically amplified by RCA. (B) Schematic overview of HybISS. Every cycle consists of hybridizing bridge-probes to RCPs and reading them out with fluorophore conjugated readout detection probes. For sequential cycles, bridge-probes are then stripped off to allow for rehybridizing next round of bridge-probes. (C) Example images of 5 cycles of a single cell. Sequential cycles with different bridge-probe libraries allows for the decoding of target transcripts within a cell. Scale bar: 10 μm.

Figure 2.

Comparison of HybISS vs SBL-based ISS. (A) Representative images on the distribution of reference genes in sequential mouse coronal sections targeted by SBH- or SBL-based chemistries. Each channel has been scaled to the same intensity for comparison. Inset displays nuclear DAPI. Scale bar: 10 μm. (B) Magnification of a single nuclei marked by DAPI with RCPs, intensities have been rescaled for clarity. Scale bar: 5 μm (C) Binned pixel intensity from images in panel (A) for each channel. Green = Cy3, blue = Cy5, red = Atto425, yellow = AF488. (D) Max intensity of top 300 RCPs from three ROIs in each channel measured, comparing SBL- and SBH-based chemistries. (E) Average intensities of measured RCPs in ROIs. Pixel intensity measured across a 21-pixel line bisecting RCPs (blue = SBH, red = SBL). (F) Calculated SNR from intensity values in panel (E), using outer 2-pixels at each end as the measurement for noise. (blue = SBH, red = SBL). (G) Ratio of SBH/SBL SNR values in several replicates across the three channels measured. (H) RCP detection with various thresholds in a 2000 × 2000 px ROI in the Cy5 channel for SBH and SBL using starfish (35) Blob Detector, indicating more RCPs found in SBH at each threshold. (I) Left: ROI of 5000 × 5000 px from SBH and SBL experiments with segmented nuclei. Middle: Example raw image of a single cell with Cy5 channel. Right: Example threshold detection (0.005) from CellProfiler and number of objects (magenta outline) detected. Scale bar: left 100 μm, middle 2 μm (J) CellProfiler Object counts for a range of thresholds in all channels from 5000 × 5000 px images in SBH and SBL chemistry. Black bar indicates optimal manual threshold and used for calculation in panel (K) (blue = SBH, red = SBL). (K) RCP count per cell from analyzed image used in (J). SBH DAPI count = 1333, SBL DAPI count = 1369.

Figure 3.

HybISS on mouse coronal brain section. (A) Whole mouse coronal section used for HybISS, ∼60 mm². Scale bar: 1 mm. (B) Representative images of HybISS using PLPs to map 119 genes over 5 cycles in section. Only first image includes counterstain for nuclei with DAPI. Scale bar: 10 μm. (C) MATLAB output of the decoding of 119 genes across entire section, each color/symbol marking a single transcript detected. Inset shows zoomed in representative image of gene marker plot mapped on DAPI image. (D) Selection of a subset of genes shown in (C) that have a distinct spatial laminar distribution within the neocortex.

Figure 4.

HybISS on human middle temporal gyrus brain sections. (A) DAPI nuclei stain of human tissue sections from middle temporal gyrus. Dashed red line demarcates the outer pial surface of tissue section. Area approx. 29 mm², 45 mm², 25 mm² top to bottom panel. Scale bar: 1 mm. (B) Representative images of the effects of lipofuscin in human brain tissue that can be treated with TLAQ, and HybISS amplification overcomes any residual background noise. Scale bar: 10 μm. (C) Magnified field of view from section in panel (a) of several cells across 5 cycles of HybISS. First cycle includes DAPI to show nuclei location. Scale bar: 5 μm. (D) Spatial distribution of decoded HybISS transcripts of 120 gene panel across the three tissue sections. Left, 1 649 212 spots; middle, 1 936 227 spots; right, 602 681 spots. (E) Kernel density estimation plots for a subset of individual gene transcripts that show distinct spatial distribution, including laminar anatomy of cortical tissue.