SCIENCEscience.org 29 OCTOBER 2021¥VOL 374 ISSUE 6567 587
20 μm
B
250 nm
Chr4
Chr7
Chr2
A
C
D
F
Slc17a7
Csf1r
Pvalb
Mfge8
Sst
Ndnf
DAPI
RNA seqFISH
ITS1 Malat1
Xist DAPI
DNA seqFISH+
Channel 1 Hyb 1
DAPI
Sequential immunofluorescence
Region 1 (hyb 1) Region 20 (hyb 20) Region 55 (hyb 55)
Region imaging (region1-60)
25-kb resolution imaging (channel 3)
Chr4 (hyb 65) Chr7 (hyb 68)
Chromosome paint imaging (Chr1-19, X)
10 μm
H4K20me3 SF3a66
Heterochromatin marker Nuclear speckle marker
Round I (Hyb1-16) Round II (hyb17-32)Round III (hyb33-48)Round IV (hyb49-64) Round V (hyb65-80)
Megabase resolution imaging (channel 1)
Reconstruction
Reconstruction
(y ) (y ) (y ) (y ) (y )
250 nm
E Whole genome (Chr1-19, X)
Cell 1135^1
5
10
15
X
Chromosomes
Chr14 (1-Mb res.) Chr14 (25-kb res.)
7.8 123.0 Mb 65.4 66.9 Mb
H I
G
(150-200 primary probes)
Target
Oligo-conjugated
primary antibody
Nucleus
ncRNA
RNA seqFISH
Sample from
mouse cortex
Tissue
section
Single
cell
Chromosome
DNA seqFISH+
Sequential
Immunofluorescence
76 RNA species
(mRNAs, introns and ncRNAs)
2,460 loci: ~1-Mb resolution
1,200 loci: 25-kb resolution
8 antibodies for
chromatin marks
Nuclear speckle
(12-50 primary probes)
RNA
(12 to 15-nt)
DNA
Readout
probe
Readout probe
Heterochromatin
Readout probe
Cell 1135
Primary
probe
Primary
probe
PrimaryPrimaryprobeprobe
1 (Pvalb)
5 (Astro)
9 (Exc)
2 (Vip)
3 (Ndnf)
4 (Sst)
6 (Micro)
7 (Endo)
8 (OPC/Oligo)
UMAP 1
UMAP 2
mRNA seqFISH clusters
2 μm 500 nm
2 μm
Fig. 1. Integrated spatial genomics in the mouse brain.(A) Schematic of
integrated RNA seqFISH, DNA seqFISH+, and sequential immunofluorescence (IF)
measurements in the mouse brain cortex. ncRNA, noncoding RNA; nt, nucleotides.
(B) Example RNA seqFISH, DNA seqFISH+, and sequential IF images in a brain
slice. The images are the maximum intensity z-projected from 1.5-mm z-slices. (Cand
D) The zoomed-in view of cell 1135 for 1-Mb resolution DNA seqFISH+ through
five rounds of barcoding with 16 pseudocolors from a single z-section in (C) and for
25-kb resolution DNA seqFISH+ through 60 hybridization rounds of adjacent region
imaging followed by 20 hybridization rounds of chromosome painting shown with
pseudocolors with spots from all z-slices in (D). White boxes on pseudocolor spots
indicate identified barcodes, and the red box indicates a rejected nonspecific binding
spot. (E) 3D reconstruction of a single nucleus colored by chromosomes (top) or
chromosome coordinates (bottom). (F) Frequencies of on- and off-target barcodes in
fluorescent channels 1 and 2 per cell. (G) Average frequencies of individual on-target
and off-target barcodes in (F). (H) Agreement between the spatial proximity maps
(probability of pairs of loci within 500 nm in cells for 1-Mb-resolution data and
within 150 nm in alleles for 25-kb-resolution data) from DNA seqFISH+ (upper right)
and normalized Hi-C read counts (lower left) at different genomic scales. Hi-C
data are displayed with 1-Mb and 25-kb bin sizes to compare with 1-Mb and 25-kb
resolution DNA seqFISH+ data, respectively. Hi-C data from mouse brain cortex
originally at 40-kb binning ( 19 ) was recomputed at 25-kb binning for direct
comparison. (I) Uniform manifold approximation and projection (UMAP)
representation of the cell type clusters determined by mRNA seqFISH profiles.
n= 2762 cells from three biological replicates in (F) to (I). Astro, astrocytes;
Micro, microglia; Endo, endothelial cells; OPC/Oligo, oligodendrocyte precursor
cells and oligodendrocytes; Exc, excitatory neurons.
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