barcoded sindbis virus  (Jackson Laboratory)

Journal: Nature Communications

Article Title: Massive multiplexing of spatially resolved single neuron projections with axonal BARseq

doi: 10.1038/s41467-024-52756-x

Figure Lengend Snippet: A Workflow. The brain is injected with barcoded viral library. After 24–48 h of expression, during which RNA barcodes are transported to axon terminals, where they are amplified into rolonies and sequenced. B , C Single rolonies in axons have significantly weaker signals compared to somatic rolonies. B Representative image of somatic and axonal rolonies; dotted circle: somatic rolonies; arrow: axonal rolonies, with zoom-in views shown on the right; rolony intensity is color coded. Scale bar: 100 µm. C Quantification of intensity between axonal and somatic rolonies. Due to the large intensity difference between somatic and axonal rolonies, proper exposure for axonal rolonies often results in saturation of somatic rolonies. Paired t -test, two-tailed, p -value < 0.0001. D Representative images of axonal and somatic rolonies in AudC/I and ipsilateral thalamus. Images are from the first cycle of in situ sequencing. Similar results are observed across sections of the same brain regions. Dotted line, anatomical boundaries. Scale bar: top, 100 µm; bottom, 25 µm. E Registered barcode signals in CCFv3. Top, data in 3D model. Gray, brain outline. Bottom, coronal view of 25 µm of the sample. Gray, DAPI. F Representative images of in situ sequencing soma and a single axonal rolony with the same barcode. Soma ROI, 30.25 µm × 30.25 µm from injection site; axonal rolony ROI, 14.85 µm × 14.85 µm from ipsilateral thalamus. In total, 17 sequencing cycles are shown. G An example of tracing tracks for a single barcoded neuron reconstructed by connecting rolonies. Rolony location is indicated in white; soma location is indicated as a large green dot in ipsilateral cortex. AudC/I, contra/ipsilateral auditory cortex; Thal, thalamus; BC, barcode; D, dorsal; V, ventral; C, caudal; R, rostral; Seq, sequencing cycle.

Article Snippet: The sindbis virus (SINV) barcode library used in this study was generated by the MAPseq core facility at Cold Spring Harbor Laboratory.

Techniques: Injection, Expressing, Amplification, Two Tailed Test, In Situ, Sequencing

Journal: bioRxiv

Article Title: Wiring logic of the early rodent olfactory system revealed by high-throughput sequencing of single neuron projections

doi: 10.1101/2021.05.12.443929

Figure Lengend Snippet: ( a ) Schematics of the MAPseq strategy which uses RNA barcodes to label neurons and map their brain-wide projections. ( b ) Infection of mitral and tufted cells by Sindbis virus carrying the barcodes and a fluorophore (GFP). ( c ) Laser Capture Micro-Dissection of target brain regions from Nissl stained coronal sections and corresponding sections registered to the Allen Brain reference atlas. ( d ) Illustration of laminar positions of mitral, tufted, and deep cells (Left) and an example BARseq sequencing image of the barcoded cells (Center). The first several bases of the barcode sequences in two example neurons analyzed via BARseq and their projection patterns across 6 bulb target brain regions (Right). Scale bar = 100 µm. ( e ) Projection patterns of neurons (415 neurons, 2 mice) identified via BARseq and their soma locations relative to the mitral cell layer (MCL). Columns represent olfactory bulb projection target regions and rows indicate individual neurons. Cell identities based on soma positions are shown on the right. Projection strength of each barcoded neuron has been normalized so that the maximum strength is 1 in each neuron (row). ( f ) (Left) Soma positions of template neurons shown relative to MCL (y-axis) and to glomerular layer (x-axis) that were used to train the projection-based classifier. Neurons are colored by their identities based on laminar positions (tufted, mitral and deep cells). (Right) The classification confusion matrix of all three classes of neurons using the BARseq-based classifier versus the position-defined classes. ( g )-( i ) The projection patterns of all MAPseq analyzed neurons ( g ), their mean projection patterns ( h ), and five example neurons ( i ) of the three classes of bulb projection neurons identified via a BARseq-based classifier. In ( g ), columns represent projection brain regions and rows indicate individual barcoded neurons. Barcoded neurons are sorted by probability of cell type classification based on running their projection patterns through the classifier. ( j ) Distribution of the broadness of projections, as measured by Inverse Participation Ratio (IPR, x-axis) at brain region-level. ( k ) Pearson correlation between putative mitral cell (pMC) projections to different target regions. Only correlations that passed statistical significance after Bonferroni correction are shown. ( l ) Distribution of the city block distance between the projection patterns of each pMC identified using the BARseq-based classifier and the most similarly projecting pMC within the same brain (blue), across different brains (red), across all brains (6) sampled after shuffling all elements in the projection matrix (yellow), or after shuffling the neuron identities for each area separately (purple).

Article Snippet: A barcoded Sindbis virus library (JK100L2, Addgene plasmid #79785) was generated and used for MAPseq and BARseq experiments as described previously – .

Techniques: Infection, Virus, Dissection, Staining, Sequencing, Blocking Assay

Journal: bioRxiv

Article Title: Wiring logic of the early rodent olfactory system revealed by high-throughput sequencing of single neuron projections

doi: 10.1101/2021.05.12.443929

Figure Lengend Snippet: ( a ) (Left) Projection patterns of piriform cortex output neurons to extra-piriform brain regions (Supplementary Table 4) and (Right) within the piriform cortex along the A-P axis. Projection density is color-coded on a log scale. In the piriform cortex, for a given barcoded neuron, the A-P position with the most barcode counts is taken as the location of the soma. ( b ) Mean projection strengths (log scale) of projections from somata at the indicated locations (x-axis) to the specific A-P positions within the piriform cortex (y-axis). ( c ) Differences in reciprocal projections between two A-P positions in the piriform cortex obtained by calculating the difference between the connectivity matrix (b) and its transpose. Blue indicates stronger projection in the posterior direction, and red indicates stronger projection in the anterior direction. ( d ) (Left) The strength of intra-piriform projections relative to their soma locations (blue). Red line indicates fit using an inverse power law model (Methods). The density of projections decreases by half at about 0.5 mm from soma location (maximum density of barcodes), making the projections width equal to 1 mm at 50% density (arrows). Contribution from dendritic neuropil of barcoded neurons was minimized by removing slices adjacent to the peak of barcode molecule counts (Methods). (Right) the same distribution obtained for pMC is substantially broader. ( e ) Mean projection patterns (Top) and the projection patterns of individual neurons (Bottom) of groups of piriform cortex output neurons to extra-piriform target brain regions. ( f ) Fraction of neurons belonging to each group at the indicated A-P positions within the piriform cortex. The color codes used are the same as in ( e ).

Article Snippet: A barcoded Sindbis virus library (JK100L2, Addgene plasmid #79785) was generated and used for MAPseq and BARseq experiments as described previously – .

Techniques:

Journal: bioRxiv

Article Title: Wiring logic of the early rodent olfactory system revealed by high-throughput sequencing of single neuron projections

doi: 10.1101/2021.05.12.443929

Figure Lengend Snippet: ( a ) Mean projection patterns of piriform cortex output neurons at the indicated A-P position of barcoded somata in the piriform cortex. Dotted lines indicate linear fits and shaded areas the range of fits from bootstrap. ( b ) Mean loadings for the first two principal components of the mean projection strengths of piriform output neurons to AON, CoA, lENT and OT sampled at the indicated A-P positions in the piriform cortex. Dotted lines indicate linear fits for APC and PPC. ( c ) Mean projection strengths of piriform projection neurons to extra-piriform target regions, organized by the location of their somata along the A-P axis of the piriform cortex (y-axis) plotted against the mean projection strengths of pMC neurons to extra-piriform bulb target regions weighted by projections to a particular A-P position in piriform cortex, P(target|PC location) (x-axis). Colors indicate A-P positions in the piriform cortex. ( d ) Cartoon schematics of the parallel olfactory circuits engaging the olfactory bulb-to-piriform inputs, piriform cortex (APC and PPC) outputs and extra-piriform bulb target regions (AON, CoA and lENT) sampled in this study.

Article Snippet: A barcoded Sindbis virus library (JK100L2, Addgene plasmid #79785) was generated and used for MAPseq and BARseq experiments as described previously – .

Techniques: