Science - USA (2021-10-29)

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.

RESEARCH | RESEARCH ARTICLES