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Advantages of Single Nucleus Over Single Cell RNA.7
Advantages of Single Nucleus Over Single Cell RNA.7
Advantages of Single Nucleus Over Single Cell RNA.7
org
ABSTRACT
Background A challenge for single-cell genomic studies in kidney and other solid overcome this limitation, it may not
tissues is generating a high-quality single-cell suspension that contains rare or dif- be feasible financially. Second, current
ficult-to-dissociate cell types and is free of both RNA degradation and artifactual enzymatic and mechanical methods for
transcriptional stress responses. single-cell dissociation introduce
Methods We compared single-cell RNA sequencing (scRNA-seq) using the DropSeq stress-induced transcriptional artifacts.
platform with single-nucleus RNA sequencing (snRNA-seq) using sNuc-DropSeq, DroNc- Although cold-active proteases have
seq, and 10X Chromium platforms on adult mouse kidney. We validated snRNA-seq on mitigated this concern in easy to disso-
fibrotic kidney from mice 14 days after unilateral ureteral obstruction (UUO) surgery. ciate embryonic kidney, 6 this has not
been shown to work in adult or diseased
Results A total of 11,391 transcriptomes were generated in the comparison phase.
kidney. Moreover, cold-active proteases
We identified ten clusters in the scRNA-seq dataset, but glomerular cell types were
still bias toward the selection of easily
absent, and one cluster consisted primarily of artifactual dissociation–induced stress
dissociated cell types. Finally, current
response genes. By contrast, snRNA-seq from all three platforms captured a diver-
approaches are incompatible with fro-
sity of kidney cell types that were not represented in the scRNA-seq dataset, in-
zen archival material, which compli-
cluding glomerular podocytes, mesangial cells, and endothelial cells. No stress
cates analysis when tissue availability
response genes were detected. Our snRNA-seq protocol yielded 20-fold more
is unpredictable or the diagnosis re-
podocytes compared with published scRNA-seq datasets (2.4% versus 0.12%, re-
quires time—such as renal biopsies.7
spectively). Unexpectedly, single-cell and single-nucleus platforms had equivalent
gene detection sensitivity. For validation, analysis of frozen day 14 UUO kidney
revealed rare juxtaglomerular cells, novel activated proximal tubule and fibroblast
METHODS
cell states, and previously unidentified tubulointerstitial signaling pathways.
Conclusions snRNA-seq achieves comparable gene detection to scRNA-seq in adult Single-Cell Dissociation and
kidney, and it also has substantial advantages, including reduced dissociation bias, Methanol Fixation
compatibility with frozen samples, elimination of dissociation-induced transcrip- Kidney from a C57BL/6 mouse was
tional stress responses, and successful performance on inflamed fibrotic kidney. minced into 1-mm pieces with a razor
J Am Soc Nephrol 30: 23–32, 2019. doi: https://doi.org/10.1681/ASN.2018090912 blade and incubated at 37°C in enzyme
single-cell RNA sequencing (scRNA- kidney cell types, because dissociation Correspondence: Dr. Benjamin D. Humphreys,
seq).1–5 The high throughput and sen- itself may damage sensitive cells while Division of Nephrology, Washington University
School of Medicine, 660 South Euclid Avenue, CB
sitivity of scRNA-seq make it well at the same time, failing to release oth- 8129, St. Louis, MO 63110. Email: humphreysbd@
suited to comprehensively map cell- ers that are surrounded by collagenous wustl.edu
state changes during disease, but three matrix. 1 Although sampling a much Copyright © 2019 by the American Society of
limitations prevent wider adoption of larger number of cells can partially Nephrology
dissociation buffer containing 250 U/ml 0.07% BSA, and 0.1% RNase inhibitor),
Significance Statement
Liberase (Roche) and 40 U/ml DNase I. filtered through a 20-mm cell strainer
After 10 minutes, the solution was trans- (43–50020–50; pluriSelect), and counted. Massively parallel single-cell RNA sequenc-
ferred to a Miltenyi C-tube, and the ing technologies provide powerful new
possibilities to understand cell complexity,
gentleMACS D1 program was run Single-Cell DropSeq,
but which platform is best suited to study
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(Miltenyi). Cells were further digested sNuc-DropSeq, DroNc-seq, adult kidney in health and disease is un-
for an additional 10 minutes with tritu- and sNuc-103 defined. The authors report that single-
ration, and the reaction was stopped by We used soft lithography techniques9 to nucleus RNA sequencing offers comparable
adding 10% FBS. The dissociated cells fabricate DropSeq and DroNc-seq sili- gene expression quantitation (despite re-
duced mRNA in the nucleus compared with
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24 Journal of the American Society of Nephrology J Am Soc Nephrol 30: 23–32, 2019
www.jasn.org RAPID COMMUNICATION
sCellDropseq sNucDropseq
A DroNcSeq sNuc-10x
B
Exonic lntronic lntergenic Exon Exon+lntron
6000 900
% of mapped reads
60 20
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4000 600
40 10
2000 300
20 0 0 0
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D eq
ro eq
sN eq
D eq
ro eq
sN eq
D eq
ro eq
sN eq
0x
0x
0x
-1
-1
-1
s
ps
cS
s
ps
cS
s
ps
cS
sN rop
sN rop
sN rop
uc
uc
uc
ro
ro
ro
N
N
0
lD
lD
lD
uc
uc
uc
D
D
el
el
el
sC
sC
sC
C Exon Exon+intron D
sCellDropseq sNucDropseq sCellDropseq sNucDropseq
20 20 Platform
DroNcSeq sNuc-10x
15 15
2500
10 10
% non-zeros
5 5 2000
0 0
DroNcSeq sNuc-10x
20 20 1500
15 15
1000
10 10
5 5
500
0 0
Cells 0 10000 20000 30000
Sequencing depth (mapped reads)
PT(S1)
PT(S2)
PT(S1-S2)
PT(S3)
PT(S3)
LH
LH
DCT
DCT-PC
CD-PC
Artifact
PT(S1)
PT(S1)
PT(S2)
PT(S2)
PT(S3)
PT(S3)
LH
LH
DCT
DCT
CD-PC
CD-PC
Artifact
Figure 1. Single nucleus RNA sequencing performs equivalent to or better than single cell RNA sequencing as long as intronic reads are
mapped. (A) Reads mapped to exonic, intronic, and intergenic regions according to the platform. (B) Average number of reads per cell
(nRead), average number of unique genes per cell (nGene), and average percentage of mitochondrial reads per cell across platforms and
according to using exonic reads only or exonic and intronic reads. (C) Percentage of nonzero reads per cell across all techniques on the basis
of exonic reads alone or exonic and intronic reads. (D) Mapped reads to a gene plot30 using different platforms. Single-cell DropSeq
J Am Soc Nephrol 30: 23–32, 2019 Single-Cell RNA Sequencing of Fibrotic Kidney 25
RAPID COMMUNICATION www.jasn.org
I J
Impact of intronic reads on DroNc-Seq clustering Impact of intronic reads on sCellDropseq clustering
Cluster separation
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PT(S3) LH
0.50 LH DCT
0.50
DCT PC
CD-PC
0.25
Read.type 0.25 Read.type
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Exon Exon
0.00 Exon+intron Exon+intron
0.00
Figure 1. Continued.
performed DropSeq. Additionally, we actually achieved superior per cell gene (Figure 1, G and H). Although scDropSeq
modified a nuclear isolation protocol11 detection than scDropSeq. The impor- yielded one more cluster than DroNc-seq,
for kidney, prepared nuclear suspen- tance of mapping both exonic and in- this was an artifact cluster expressing
sions (Supplemental Figure 1) from tronic reads for improved quality of the stress response genes induced during dis-
snap-frozen kidney that had been stored snRNA-seq datasets is illustrated by the sociation. Including intronic reads im-
for 1 week at 280°C, and performed improvement in the percentage of non- proved cluster cohesion (the average
single-nucleus RNA sequencing (snRNA- zero reads across all three platforms (Fig- within cluster coclustering) and separa-
seq). A total of 11,391 transcriptomes were ure 1C). At lower mapped read depths, tion (the average coclustering difference
generated. Figure 1A shows that, for both scDropSeq and DroNc-seq detected with the nearest cluster) for DroNc-seq
single-cell DropSeq (scDropSeq), a ma- 10%–25% more unique genes per cell but not scDropSeq (Figure 1, I and J).13
jority of mapped reads were exonic, than snDropSeq or sn103; however, Ambient mRNA released during tis-
whereas for single-nucleus DropSeq the difference narrowed at higher read sue dissociation can contaminate
(snDropSeq), DroNc-seq, and sn103, a depths (Figure 1D). scRNA-seq data. For example, highly ex-
majority of reads where intronic. The av- We compared unsupervised clustering pressed genes (for example, Slc34a1)
erage number of mapped reads was simi- results using a matched set of epithelial from the highly abundant proximal tu-
lar across platforms but only if both exon cell and nucleus transcriptomes. bule can be detected in all cell clusters in a
and intron reads were included (Figure Using the tubular epithelial cells common recent dataset 2 (Supplemental Figure
1B). Surprisingly, the average number of to our scDropSeq and DroNc-seq datasets, 2A). We found similar tubular contami-
genes detected per cell was also compara- we selected the 1469 best-matching cell- nation across platforms in our data, al-
ble across all four platforms as long as nucleus pairs by calculating dropout though the degree of contamination was
we used both mapped exon and intron weighted Pearson correlations as de- somewhat reduced in snRNA-seq com-
reads, although nuclei contain substan- scribed. 13 Using exonic reads alone, pared with scRNA-seq (Supplemental
tially less mRNA than whole cells (Figure we could identify only four clusters Figure 2, B–E).
1B). Because mitochondria are cytosolic, from the DroNc-seq dataset, whereas We used integrated analysis to identify
we only detected mitochondrial tran- the scDropSeq dataset yielded seven conserved cell types generated using dif-
scripts using scDropSeq, where they clusters (Figure 1, E and F). The inclu- ferent platforms (scRNA-seq and
made up 24% of all scDropSeq genes (Fig- sion of introns improved DroNc-seq snRNA-seq techniques) after batch cor-
ure 1B). After subtracting mitochondrial clusters to six cell types but did not change rection with aligned canonical correla-
genes, all three single-nucleus techniques the number of clusters for scDropSeq tion analysis.14 We identified 13 clusters
(scDropSeq) and DroNc-seq show an advantage in the low- (10,000 mapped reads per cell) to middle-range (20,000 mapped reads per cell)
sequencing depths. (E) The t-distributed stochastic neighbor embedding (tSNE) plot of 1469 epithelial cells from the DroNc-seq dataset
on the basis of mapped exonic reads alone. (F) tSNE of 1469 matched epithelial cells from the scDropSeq dataset (also on the basis of
exonic reads alone). (G) Improved clustering from 1469 epithelial cells from the DroNc-seq dataset using exonic plus intronic reads. (H)
Few changes in clustering of 1469 matched epithelial cells from the scDropSeq dataset using exonic plus intronic reads. Cluster cohesion
(average within-cluster coclustering) and separation (difference between within-cluster coclustering and maximum between-cluster co-
clustering)13 plotted for (I) nuclei and (J) cells. Gene expression quantification, including introns, increases cohesion and separation of
nuclei but not cell clusters. CD-PC, collecting duct-principal cell; DCT, distal convoluted tubule; LH, loop of Henle; PT, proximal tubule.
26 Journal of the American Society of Nephrology J Am Soc Nephrol 30: 23–32, 2019
www.jasn.org RAPID COMMUNICATION
A B Avg.Exp
Pod Pct.Exp 25 50 75 100
0.0
0.5
1.0
1.5
2.0
2.5
M Pod Pod
MC MC
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S3 EC EC
MC PT(S1-S2)
PT(S1-S2)
PT(S3)
LH(DL) LH(DL) PT(S3)
S1-S2 LH(AL) LH(DL)
DCT
EC LH(AL)
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PC CNT
CD-PC DCT
CNT IC-A CNT
LH(AL) IC-B
DCT CD-PC
M
IC-A
IC-B IC-B
IC-A M
t
s1 hl2 cn a12 a13 ha7 2a1 2a3 8a1 qp2 Ki 6a4 tprc
ph F Em 5 7 p 1 1 lc A
c lc E lc lc S c2 P
N l l
S S S S S
C D E
sCellDropseq sCellDropseq DroNcSeq p=0.016
sNucDropseq sNucDropseq TenxNuc 3
DroNcSeq
TenxNuc
4 Technique
scRNAseq
3 snRNAseq
1
2 Dataset
Park et al
1 This study
0
0
Figure 2. Reduced dissociation bias from single-nucleus techniques. (A) The t-distributed stochastic neighbor embedding (tSNE) pro-
jection of the combined datasets reveals 13 separate clusters. CD-PC, collecting duct-principal cell; CNT, connecting tubule; DCT, distal
convoluted tubule; EC, endothelial cell; IC-A, intercalated cell type A; IC-B, intercalated cell type B; LH(AL), loop of Henle ascending loop;
LH(DL), loop of Henle descending loop; MF, macrophage; MC, mesangial cell; Pod, podocyte; PT, proximal tubule. (B) Marker gene
expression across clusters for the combined dataset. (C) tSNE showing the contribution of data from each platform to all clusters. (D)
Percentage of cells contributed by each platform reveals a very low contribution to podocytes, endothelial cells, and intercalated cells
type A and type B from single-cell DropSeq (scDropSeq) compared with single-nucleus platforms. (E) We combined podocyte frequencies
obtained from our scDropSeq (n=1) as well as those from Park et al.2 (n=7) and compared them with the frequencies observed in our single-
nucleus RNA sequencing (snRNA-seq) datasets (n=3). This revealed 20-fold more podocytes from snRNA-seq (2.4%) compared with
single-cell RNA sequencing (scRNA-seq; 0.12%; P=0.02).
in the combined dataset, including po- better sensitivity to detect podocytes, more podocytes from snRNA-seq com-
docytes, endothelial cells and mesan- endothelial cells, and intercalated cells pared with scRNA-seq (2.4% versus
gium, nine tubule clusters, and one (Figure 2D). In fact, when the scDropSeq 0.12%; P=0.02) (Figure 2E).
macrophage cluster (Figure 2, A and B, data were clustered separately, there were We quantitated differential gene de-
Supplemental Material). Comparison of no independent podocyte or endothelial tection in cells versus nuclei and asked
this dataset with three other recent kid- cell clusters at all, whereas all three single- how this might influence interpretation
ney RNA sequencing datasets confirmed nucleus platforms identified these cell of results. 16 Following a recently de-
our cluster annotations (Supplemental types (Supplemental Figure 4). We com- scribed analytic approach,13 we found
Figure 3).2,3,15 Projecting the source of bined podocyte frequencies obtained that a majority of expressed genes
each cell onto the tSNE revealed the from our scDropSeq as well as those (9588; 71.4%) showed similar detection
relative contribution of each plat- from Park et al.2 and compared them (,20% difference) in nuclei and cells,
form to each cluster (Figure 2C). All with the frequencies observed in our whereas only 452 genes (3.4%) were de-
three nuclear approaches had much snRNA-seq datasets. This revealed 20-fold tected in at least 25% more cells than
J Am Soc Nephrol 30: 23–32, 2019 Single-Cell RNA Sequencing of Fibrotic Kidney 27
RAPID COMMUNICATION www.jasn.org
A B
5.0% Cells > Nuclei 6.4% Nuclei > Cells
1.00 300
Cellular proportion
1000 200
1000
0.50 100
100
10 10
100
1 1
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0.25
0.00 0
tSNE_2
Pod
Normalized UMI count
0 0 0 MC
mt-Cytb Rpl22 Hspa8 Slc22a23 Foxp1 Gm26804
8 -10 EC
6
4 6
4 -20
2 2 -30
0 0
mt-Nd1 Rps27l Hspd1 -20 0 20 40
Slc16a10 Mecom Malat1
6 8 tSNE_1
4 6
4
2
2
0 0
F G H
AUROC
30
0
0.4
0.8
PT(S1)
20
PT(S2)
10 Platform sCell_MC PT(S3)
tSNE_2
sNuc_MC
sCell LH
0 sCell_Pod
sNuc DCT
sNuc_Pod
-10 CD-PC
sNuc_EC
-20 sCell_EC Artifact
EC
uc C
sC MC
C
el d
sN Pod
-30
o
_E
M
_P
l_
l_
l_
uc
uc
el
el
-20 0 20 40
sC
sN
sC
sN
tSNE_1
Figure 3. Single nucleus RNA-seq detects similar genes to single cell RNA-seq without artifactual transcriptional stress responses. (A) Binned
scatterplot showing the proportion of genes detected with greater reliability in cells versus nuclei. The gray lines show the variation in detection
expected by chance (95% confidence interval). (B) Binned scatterplot showing that 5.0% of genes are significantly more highly expressed (fold
change .1.5; adjusted P value ,0.05) in cells and that 6.4% of genes are significantly more highly expressed in nuclei. (C) Cell-enriched genes
include mitochondrial and ribosomal genes as well as heat shock response genes. (D) Nuclei-enriched genes predominantly encode drivers of cell
identity, such as solute carriers, transcription factors, and long noncoding RNA. (E) The 650 glomerular cells from DroNc-seq and single-nucleus
DropSeq (snDropSeq) plus the 650 matched cells from a glomerular cell atlas3 coprojected by the t-distributed stochastic neighbor embedding
(tSNE) reveal podocyte (Pod), mesangial cell (MC), and endothelial cell (EC) clusters. (F) Equal representation of cell and nucleus RNA sequencing
data in all clusters. (G) Strong replicability of glomerular cell types between cell and nucleus datasets as defined by the area under the receiver
operator characteristic curve (AUROC) score.18 (H) tSNE of epithelia from single-cell DropSeq (scDropSeq) highlighting an artifactual cluster
defined by stress response gene expression induced during proteolytic dissociation. CD-PC, collecting duct-principal cell; DCT, distal convoluted
tubule; LH, loop of Henle; PT, proximal tubule. (I) Immediate early gene expression in the artifactual cluster. (J) Reanalysis of the glomerular cell
atlas3 reveals strong stress response gene expression among podocytes, mesangial cells, and endothelial cells. The same cells isolated by nuclear
dissociation lack a stress response signature. (K) Heat map comparison of the same glomerular cell types showing strong mitochondria, heat shock,
and apoptosis gene expression signature among the single-cell but not the single-nucleus dataset. FC, fold change; TF, transcription factor; UMI,
unique molecular identifier.
28 Journal of the American Society of Nephrology J Am Soc Nephrol 30: 23–32, 2019
www.jasn.org RAPID COMMUNICATION
I J
Gadd45b Egr1 Nr4a1 Atf3 Fosb Fos Jund Junb Jun
Fos Fosb
0 2 0 2 0 2 0 2 0 2 0 2 0 2 0 2 0 2
Pod
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sCell
Jun Jund
MC
EC
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Pod
sNucleus
MC
EC
K
Mitochondria Heat shock Apoptosis
Geneset
2 Geneset
Pod
Mitochondrion
1 Heat shock response
MC
Apoptosis
sCell
0
Platform
sCell_Pod
–1
sCell_MC
EC
sCell_EC
sNuc_Pod
sNuc_MC
Pod
sNuc_EC
sNucleus
MC
EC
Platform
mt.Cytb
mt.Nd1
mt.Rnr2
mt.Nd2
mt.Nd4
mt.Nd6
mt.Nd5
mt.Rnr1
mt.Co1
Hspa1a
Hsp90ab1
Hspa1b
Hspb1
Hspa8
Hspa5
Hsp90b1
Dnaja1
Tpt1
Jun
Junb
Jund
Ubb
Ubc
Lmna
Ier3
Dynll1
Ppp1r15a
Crip1
Atf3
Figure 3. Continued.
nuclei, and 266 genes (2.0%) were de- with a recent report from the brain.13 We Supplemental Figures 5) with equivalent
tected in at least 25% more nuclei than could also detect long noncoding RNAs contributions to each from the cell
cells (Figure 3A). Similarly, only 5.0% preferentially in nucleus compared with and nucleus datasets (Figure 3F). Using
(676 genes) of detected genes were ex- whole cell (Figure 3D).16 MetaNeighbor, we validated that each
pressed more highly in cells than in nu- We next asked whether these differ- glomerular cell type identified by
clei (fold change .1.5; adjusted P value ences might alter cell classification scDropSeq had a very high area under
,0.05), and 863 genes (6.4%) were ex- using a recently published mouse glo- the receiver operator characteristic curve
pressed more highly in nuclei than in merular single-cell atlas generated using score for the corresponding cell type
cells (Figure 3B). Examples of genes en- DropSeq.3 We extracted podocytes, en- identified by snDropSeq and very low
riched in the scDropSeq dataset included dothelial cells, and mesangial cells (650 area under the receiver operator charac-
mitochondrial and ribosomal genes as cells total) from our snDropSeq and teristic curve scores for the other two cell
well as genes in the heat shock pathway DroNc-seq datasets and used a random types (Figure 3G).18 This indicates that
(Figure 3C). Surprisingly, nucleus- forest model to choose the 650 best- our snRNA-seq dataset replicates cell
enriched genes included many genes matching cells from the glomerular cell classification with a high degree of con-
that drive cell identity, such as solute car- atlas.17 The combined datasets clustered fidence, despite differences in abundance
riers and transcription factors, consistent into three distinct cell types (Figure 3E, of some genes in nuclei versus whole cell.
J Am Soc Nephrol 30: 23–32, 2019 Single-Cell RNA Sequencing of Fibrotic Kidney 29
RAPID COMMUNICATION www.jasn.org
A E
UUO day 14 Mrc2
M 5
4
Pod 3
EC 2
PC 1
S1 PT(S1)
S3 0
PT(S2) Ednra
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S2 PT(S3) 5
4
Pod 4
JGA CNT
TAL 3
CD-PC 2
Fib.1 IC 1
Act. Fib1 0
JGA Akap12
5
IC Act. Fib2 4
M 3
2
EC 1
0
b1
Ac GA
b2
Fi
Fi
J
t.
t.
Ac
B C
p6 b
45
C 0d2
At 45
Cell cycle
2
Sl a1
Pd a3
ap
t2
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C 1
a1
a1
s2
Sl 7
b
a
em
am
v
2
1
ga
ha
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p2
hg
2
c5
c5
c1
c1
ph
en
av
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c
fh
cl
Tm
B3
Ep
To
Tn
Ar
Sl
Sl
Fl
N
R
Pod
EC
Phase PT(S1)
G1
G2M PT(S2)
S PT(S3)
Dedif. PT
Prolif. PT
DL + tAL
TAL
DCT
D Dock10
CNT
CD-PC
IC
Act. Fib1
gene JGA
3.5 Act. Fib2
3.0
2.5 M
2.0
F Ligands Receptors G
Gpc3 Tubulointerstitial crosstalk
Igf1r
Bmp6 Gpc4
Lama1 Slit3
Vcam1 Lrp2
Cd36 Sema3d
Icam1
Adam12 Plxna2 Ccl2 Pdgfd
Sema6a Itga1 IL-34
Sema3c Itgav Cxcl1 Fib
Col18a1 Nt5e Cxcl2
Vim Itgb8
Plat Bmp6 p1 Cf
Itgb6 Sp h
Serpine1 Gpc3
Hspg2 Cd44 Serpine1 Ncam1
Lama4 Acvr1 PT
Itga3 Tnc
Col14a1 ApoE
M
Slit3 Nrp2
Sema3d Icam1
Plxna4
Col8a1 Ptprb
Col5a2 Se Hspg2
Sdc2 m
Lama2 Itgb5 Se a3
c
Col1a2 m
Robo1 a6
Col3a1 a
Cfh Pdgfrb
Pdgfd Cacna1c EC
Spp1 Ddr2
Col4a1 Itga9
Fn1 Nrp1
Col4a6 Robo2
Ncam1 Chrm3
Tnc
Apoe Itgam
er PT (S1)
ol tia (S )
ra d )
Ac ting PT
PT
at F C
Fi 1
b2
Pr en T 2
ife te 3
ed ib
M
tiv ted E
( )
ol tia (S )
ra d )
Ac ting PT
PT
tiv ted EC
Fi 1
b2
P (S
PT (S1
Pr ren PT S2
ife te 3
ed ib
M
at F
PT
PT
Activa
Ac a
tiv
-2 -1 0 1 2
iff
e
iff
ed
ed
Z score
D
D
30 Journal of the American Society of Nephrology J Am Soc Nephrol 30: 23–32, 2019
www.jasn.org RAPID COMMUNICATION
Stress response genes are induced but not others (Havcr1), and also, it ex- trate how snRNA-seq can reveal unex-
during proteolytic tissue dissociation at pressed many secreted proinflammatory pected intercellular communication
37°C.6 In our scDropSeq dataset, an en- cytokines, including the macrophage pathways. We uncovered known (Bmp6,
tirely new cluster was formed on the chemoattractant Ccl2, 19 the macro- Pdgfd, and Spp1) and novel (Sema3c,
basis of stress response genes (Figure 3, phage proliferative cytokine Il34,20 and Sema6a, Gpc, and Slit3) signaling relation-
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H and I). Nuclear dissociation is carried the neutrophil chemoattractants Cxcl1 ships (Figure 4F). Figure 4G summarizes
out on ice, preventing new gene tran- and Cxcl2.21 Differential gene expres- these fibrotic kidney tubulointerstitial
scription. We could detect abundant sion between these two novel proximal crosstalk pathways.
stress response gene expression in all tubule cell states showed that the prolif- In summary, snRNA-seq provides re-
nYQp/IlQrHD3i3D0OdRyi7TvSFl4Cf3VC1y0abggQZXdtwnfKZBYtws= on 05/01/2023
cells from the mouse glomerular atlas, erating cluster was dominated by cell duced dissociation bias and equivalent
which was absent from data generated cycle gene ontology terms, whereas the gene detection compared with scRNA-
by snDropSeq (Figure 3J, Supplemental dedifferentiated cluster was character- seq. This is important, because single-cell
Figure 7). Comparison of differential ized primarily by regulation of cell transcriptomics is costly—minimizing cell
gene expression among glomerular cell movement (Supplemental Figure 7). As number while maximizing cell representa-
types showed that mitochondrial genes, an example, the rhoGEF Dock10 was en- tion will reduce expenses. We show that,
heat shock genes, and genes associated riched in the dedifferentiated cluster although snRNA-seq enriches for a smaller
with apoptosis were detected in scDrop- (Figure 4D), and this gene regulates cell proportion (,7%) of different genes than
Seq data but absent from snDropSeq morphogenesis via the Cdc42 pathway.22 scRNA-seq, many of these are either mito-
data (Figure 3K). We could detect rare juxtaglomerular chondrial or artifactual stress response
We validated our snRNA-seq protocol apparatus cells on the basis of expression genes, and cell identification is not im-
on fibrotic and inflamed UUO day 14 of Ren1 as well as the endothelin A paired. Finally, we have used our snRNA-
kidney; 6147 single-nucleus transcrip- receptor (Figure 4E). Intriguingly, juxta- seq protocol on an inflamed fibrotic kidney
tomes were generated on the sn103 glomerular apparatus cells also ex- to illustrate novel cell states and a rare cell
platform, with an average of 763 unique pressed Hopx, a marker of adult stem type, and with our intercellular communi-
genes and 1206 unique molecular iden- cells in the intestine, brain, and hair fol- cation map, we take an initial step in
tifiers per nucleus. Unsupervised analy- licle.23–25 Renin lineage cells have been addressing a major challenge for kidney
sis identified 17 unique cell clusters by proposed to serve as podocyte progen- single-cell technologies: to synthesize these
tSNE (Figure 4A, Supplemental Mate- tors.26 We also identified two distinct ac- rich datasets with spatial information.
rial). These included two novel proximal tivated fibroblast populations. One of
tubule populations, one of which was these expressed mannose receptor 2,
characterized by a strong proliferative which binds and internalizes collagen
gene signature (Figure 4B); therefore, and attenuates renal fibrosis (Figure ACKNOWLEDGMENTS
we annotated this cluster as proliferating 4E). 27 The other activated fibroblast
proximal tubule cells. It also expressed cell type expressed tenascin C, which Primary support for this work was from grant
injury markers, including Havcr1 and has been recently identified as an extra- 173970 from the Chan Zuckerberg Initiative.
Vcam1 (Figure 4C). The other novel cellular matrix glycoprotein that pro- Additional support was from National Insti-
proximal tubule cluster was not cycling motes renal fibrosis. 28 Both of these tutes of Health/National Institute of Diabetes
but expressed a strong cell movement cell types expressed a-smooth muscle and Digestive and Kidney Diseases (NIDDK)
transcriptional signature. We annotated actin, suggesting that they are distinct grants DK103740 (to B.D.H.) and DK107374
this as a dedifferentiated proximal tubule subsets of renal myofibroblasts.29 (to B.D.H.) and NIDDK Diabetic Complica-
cluster. Intriguingly, this cluster ex- Finally, we analyzed receptor-ligand tions Consortium (www.diacomp.org) grants
pressed some injury markers (Vcam1) pairs in a cell-specific manner to illus- DK076169 and DK115255.
Figure 4. snRNA-seq of day 14 unilateral ureteral obstruction (UUO) kidney identifies rare cell types and and intercellular communication
networks. (A, inset) Periodic acid–Schiff stain of UUO kidney showing dilated and cast-filled tubules and expanded and fibrotic interstitium. (A)
The t-distributed stochastic neighbor embedding (tSNE) shows 17 separate cell clusters. (B) Projection of cell cycle state onto the tSNE, revealing
limited proliferation primarily in the proliferating proximal tubule cluster. (C) Violin plot showing cluster-specific gene expression. (D) Dock10
expression through the proximal tubule but enriched within the dedifferentiating proximal tubule cluster. (E) Three stromal clusters could be
identified, including juxtaglomerular apparatus cells expressing Endra and the stem cell marker Hopx. Immunohistochemistry images are from
the Human Protein Atlas (https://www.proteinatlas.org/). (F) Cell-specific ligand-receptor analysis reveals intercellular signaling pathways. (G)
Known and new intercellular signaling within the tubulintersitial compartment as revealed by this snRNA-seq analysis. Act., activating; CD-PC,
collecting duct-principal cell; CNT, connecting tubule; DCT, distal convoluted tubule; Dediff., dedifferentiated; DL + tAL, descending limb + thin
ascending limb; EC, endothelial cell; Fib., fibroblast; IC, intercalated cell; JGA, juxtaglomerular apparatus; MF, macrophage; PC, principal cell;
Pod, podocyte; Prolif, proliferating; PT, proximal tubule; TAL, thick ascending limb; UMI, unique molecular identifier.
J Am Soc Nephrol 30: 23–32, 2019 Single-Cell RNA Sequencing of Fibrotic Kidney 31
RAPID COMMUNICATION www.jasn.org
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