Rabies virus-based barcoded neuroanatomy resolved by single-cell RNA and in situ sequencing
- Allen Institute for Brain Science, United States
- McGovern Institute for Brain Research, Massachusetts Institute of Technology, United States
- Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Broad Institute of MIT and Harvard, United States
- Broad Institute of MIT and Harvard, United States
Figures
Figure 1
Models of multiplexed retrograde labeling and monosynaptic tracing using barcoded rabies virus.
(A) In multiplexed retrograde labeling, rabies viruses carrying different barcodes are injected into different brain regions, and retrogradely labeled neurons can be distinguished based on the …
Figure 2 with 3 supplements
High-quality cell typing of rabies virus-barcoded neurons using single-cell RNA-seq.
(A) Illustration of the design of barcoded rabies virus libraries. (B) Barcode distribution in the CCS and non-CCS libraries. The y axis indicates the count of barcodes, which are sorted in …
Figure 2—figure supplement 1
Quality control of scRNA-seq in rabies virus-infected cells.
(A) Quality control plots of scRNA-seq for two animals used in multiplexed retrograde labeling experiments (591123 and 620569) and four animals used in barcoded transsynaptic labeling experiments …
Figure 2—figure supplement 2
The expression of immune response-related genes in rabies virus-infected cells.
(A) Volcano plots showing differential expression of genes in clusters with more than 10 rabies infected cells across all six animals. X axes indicate and y axes indicate . Red/blue dots indicate …
Figure 2—figure supplement 3
The expression of activity-related genes in rabies virus-infected cells and uninfected cells from Tasic et al., 2018.
Dot size indicates the proportion of cells with nonzero marker expression, and colors indicate relative gene expression levels scaled per row.
Figure 3
In situ sequencing identifies transcriptomic types of neurons infected with barcoded rabies virus.
(A) Illustration of probe designs and amplification approach for in situ sequencing of rabies barcodes. (B) Illustration of the experiments. The two libraries were injected in VISal and RSP/SC, and …
Figure 4
Multiplexed retrograde labeling recapitulates known cortical projections.
(A) Histograms showing the distribution of the minimum Hamming distance between each barcode and all other barcodes for barcodes in the VISal library (blue), the SC/RSP library (red), and random …
Figure 5 with 3 supplements
Multiplexed transsynaptic labeling by sequencing rabies barcodes in situ.
(A) Five possible types of barcode-sharing networks in a barcoded transsynaptic tracing experiment using rabies virus. Whether each network is compatible with monosynaptic tracing and/or mapping …
Figure 5—figure supplement 1
scRNA-seq is insufficient to resolve connectivity among transsynaptically labeled neurons using barcoded rabies virus.
(A) Outline of the trans-synaptic labeling experiment using scRNA-seq. In Cre-expressing animals, we sequentially injected AV helpers (day 1) and barcoded rabies virus (day 14), then dissected VISp …
Figure 5—figure supplement 2
Barcoded transsynaptic labeling resolved by in situ sequencing.
(A) For barcoded cells with the indicated barcode complexity (x axis, left) or barcode counts per cell (x axis, right), two proofreaders manually determined whether each cell is a real barcoded cell …
Figure 5—figure supplement 3
Presynaptic cells and source cells in all single-source networks.
In each plot, a source cell (red cross) and presynaptic cells (dots) that shared the same barcodes were plotted. Colors of dots indicate transcriptomic types of presynaptic neurons. Transcriptomic …
Tables
Table 1
Number of cells in the scRNA-seq-based transsynaptic tracing and retrograde tracing experiments.
| Animal ID | N cells | After QC (total reads, genes detected, and GC content) | Mapping confidence > 0.7 | Mapping correlation > 0.6 | |
|---|---|---|---|---|---|
| Transsynaptic tracing | 591121 | 94 | 80 | 77 | 75 |
| 618308 | 80 | 71 | 69 | 64 | |
| 618309 | 60 | 46 | 45 | 42 | |
| 620588 | 61 | 55 | 53 | 51 | |
| Retrograde tracing | 591123 | 48 | 34 | 30 | 21 |
| 620569 | 48 | 41 | 40 | 33 | |
| Total | 443 | 371 | 354 | 295 |
Table 2
The numbers of barcoded cells that belonged to each type of network in the in situ sequencing-based trans-synaptic tracing experiment.
| Total | Single-source | Double-labeled | Connected-source | No-source | Lost-source | |
|---|---|---|---|---|---|---|
| Source cells and barcodes | 120 cells, 59 barcodes, 126 cell-barcode pairs | 42 cells, 43 barcodes, 43 cell-barcode pairs | Est. 33 cell-barcode pairs | Est. 50 cell-barcode pairs | 0 | 0 |
| presynaptic cells | 2590 cells (=381 + 979–6+677 + 566–7) | 381 cells | 979 cells (6 cells also contained a single-source barcode) | 677 cells (7 cells also had a lost-source barcode) | 566 cells (7 cells also had a no-source barcode) | |
| Barcodes in presynaptic cells | 535 barcodes (=31 + 16+427 + 61) | 31 barcodes | 16 barcodes | 427 | 61 | |
| Filtered out cells | 204 cells (with one G transcript and/or low-quality source cells) | NA | NA | NA | NA | NA |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Mus musculus) | C57BL/6 J (See Supplementary file 1 for details) | Jackson Laboratory | 000664 | |
| Recombinant DNA reagent | pRVdG-4mCherry (plasmid) | Weible et al., 2010 | Addgene_52488 | |
| Recombinant DNA reagent | RabV CVS-N2c(deltaG)-mCherry (plasmid) | Reardon et al., 2016 | Addgene_73464 | |
| Recombinant DNA reagent | pCAG-B19N (plasmid) | Chatterjee et al., 2018 | Addgene_59924 | |
| Recombinant DNA reagent | pCAG-B19P (plasmid) | Chatterjee et al., 2018 | Addgene_59925 | |
| Recombinant DNA reagent | pCAG-B19G (plasmid) | Chatterjee et al., 2018 | Addgene_59921 | |
| Recombinant DNA reagent | pCAG-B19L (plasmid) | Chatterjee et al., 2018 | Addgene_59922 | |
| Recombinant DNA reagent | pCAG-T7pol (plasmid) | Chatterjee et al., 2018 | Addgene_59926 | |
| Recombinant DNA reagent | pCAG-N2cN (plasmid) | This paper | Addgene_100801 | Used in rabies virus rescue (see Materials and methods M5) |
| Recombinant DNA reagent | pCAG N2cP (plasmid) | This paper | Addgene_100808 | Used in rabies virus rescue (see Materials and methods M5) |
| Recombinant DNA reagent | pCAG-N2cG (plasmid) | This paper | Addgene_100811 | Used in rabies virus rescue (see Materials and methods M5) |
| Recombinant DNA reagent | pCAG-N2cL (plasmid) | This paper | Addgene_100812 | Used in rabies virus rescue (see Materials and methods M5) |
| Sequence-based reagent | See Supplementary file 3 for details | Integrated DNA Technologies | NA | |
| Commercial assay or kit | MiSeq Reagent Nano Kit v2 (300-cycles) | Illumina | MS-103–1001 | |
| Commercial assay or kit | RevertAid Reverse Transcriptase | Thermo Fisher | EP0442 | |
| Commercial assay or kit | RiboLock RNase Inhibitor | Thermo Fisher | EO0384 | |
| Commercial assay or kit | Phusion High-Fidelity DNA Polymerase | Thermo Fisher | F530L | |
| Commercial assay or kit | Ampligase Thermostable DNA Ligase | Biosearch Technologies | A0110K | |
| Commercial assay or kit | RNase H | Qiagen | Y9220L | |
| Commercial assay or kit | Phi29 dna polymerase | Thermo Fisher | EP0094 | |
| Chemical compound, drug | Iodoacetamide, No-Weigh | Thermo Fisher | A39271 | |
| Chemical compound, drug | Bis-PEG9-NHS ester | BroadPharm | BP-21504 | |
| Software, algorithm | MATLAB | Mathworks | RRID: SCR_001622 | |
| Software, algorithm | R | R Project for Statistical Computing | RRID: SCR_001905 | |
| Software, algorithm | Custom scripts for data analysis/processing | This paper | See M15. Data and Code availability for links | |
| Other | AAV2-retro-syn-mCre (virus) | Jin et al., 2023b | Addgene_178515 (genome plasmid) | Helper virus for barcoded rabies virus (see Figure 5B) |
| Other | AAV1-syn-FLEX-splitTVA-EGFP-tTA (virus) | Liu et al., 2017 | Addgene_100798 (genome plasmid) | Helper virus for barcoded rabies virus (see Figure 5B) |
| Other | AAV1-TREtight-mTagBFP2-B19G (virus) | Liu et al., 2017 | Addgene_100799 (genome plasmid) | Helper virus for barcoded rabies virus (see Figure 5B) |
| Other | AAV1-TREtight-mTagBFP2-N2cG (virus) | This paper | Addgene_192838 (genome plasmid) | Helper virus for CVS-N2c strain of rabies virus (see Figure 5—figure supplement 1) |
| Other | N2c∆G-4mCherry_CCS2_20nt_HM(EnvA) (virus) | This paper | Barcoded rabies virus library used for monosynaptic tracing with scRNA-seq (see Materials and methods M5) | |
| Other | RV∆G-4mCherry_CCS2_20nt_HM(EnvA) (virus) | This paper | Barcoded rabies virus library used for monosynaptic tracing with BARseq (see Materials and methods M5) | |
| Other | RV∆G-4mCherry_CCS2_20nt_HM(B19G) (virus) | This paper | Barcoded rabies virus library used for retrograde labeling (see Materials and methods M5) | |
| Other | RV∆G-4mCherry_20-mer barcode(B19G) (virus) | This paper | Barcoded rabies virus library used for retrograde labeling (see Materials and methods M5) |
Table 3
List of filters and lasers used for in situ sequencing.
| Filters | ||
|---|---|---|
| Main dichroic | Filter names | |
| D1 | FF421/491/567/659/776-Di01 (Semrock) | |
| D2 | ZT405/514/635rpc | |
| D3 | FF421/491/572-Di01−25x36(Semrock) | |
| Emission filters | ||
| E1 | FF01-441/511/593/684/817(Semrock) | |
| E2 | FF01-565/24(Semrock) | |
| E4 | FF01-676/29(Semrock) | |
| E5 | FF01-775/140(Semrock) | |
| E7 | 69,401 m | |
| E8 | ZET532/640 m | |
| Imaging settings | ||
| Sequencing cycles | ||
| Channel | Filter combinations | laser |
| G | D2/E2 | 520 |
| T | D1/E1 | 546 |
| A | D2/E4 | 638 |
| C | D2/E5 | 638 |
| DIC | D2/E5 | DIA |
| Hybridization cycle | ||
| GFP | D3/E7 | 477 |
| YFP | D2/E2 | 520 |
| TxRed | D3/E7 | 546 |
| Cy5 | D2/E8 | 638 |
| DAPI | D1/E7 | 405 |
| DIC | D3/E7 | DIA |
Additional files
-
Supplementary file 1
Metadata of animals used in this study.
- https://cdn.elifesciences.org/articles/87866/elife-87866-supp1-v2.xlsx
-
Supplementary file 2
Viruses used in this study.
- https://cdn.elifesciences.org/articles/87866/elife-87866-supp2-v2.xlsx
-
Supplementary file 3
Gene panel and list of oligos used.
- https://cdn.elifesciences.org/articles/87866/elife-87866-supp3-v2.xlsx
-
MDAR checklist
- https://cdn.elifesciences.org/articles/87866/elife-87866-mdarchecklist1-v2.pdf
