Cell type composition and circuit organization of clonally related excitatory neurons in the juvenile mouse neocortex
- Department of Neuroscience, Baylor College of Medicine, United States
- Center for Neuroscience and Artificial Intelligence, Baylor College of Medicine, United States
- Department of Anatomic Pathology, University of California San Francisco, United States
- Institute for Ophthalmic Research, University of Tübingen, Germany
- Department of Computer Science, University of Tübingen, Germany
- Interfaculty Institute for Biomedical Informatics, University of Tübingen, Germany
- Department of Cell and Molecular Biology, Karolinska Institutet, Sweden
- Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, United States
- Jan and Dan Duncan Neurological Research Institute at Texas Children's Hospital, United States
- Department of Electrical and Computer Engineering, Rice University, United States
Figures
Figure 1 with 2 supplements
Tamoxifen induction at E10.5 generates translaminar clones spanning cortical layers 2–6.
(A) Schematic of tamoxifen-inducible Cre-loxP system for lineage tracing. (B) Manual reconstruction of a clone across multiple slices. In this example, larger red spots are morphologically consistent with glial cells at high magnification. Scale bar: 100 μm. (C) Examples of reconstructed clones labeled at E9.5, E10.5 or E11.5. Scale bars: 100 μm. Differences in thickness are due to tangential sectioning of the cortex rostrally (approximate rostrocaudal position of each clone is shown in gray beneath the reconstruction). (D and E) Number of neurons (D) and clone width (E) at postnatal day 10 following tamoxifen induction at E9.5, E10.5, or E11.5 (n = 35, 39, and 50 clones; n = 3, 4 and 3 mice per condition; p-values computed using Wilcoxon rank sum). (F) Percent of clones that are do not contain neurons in L5 or L6 following tamoxifen induction at E9.5, E10.5, or E11.5 (n = 0/35, 1/39, and 10/50 clones; n = 3, 4 and 3 mice per condition; p-values computed using Fisher’s exact test). Error bars show 95% Clopper-Pearson confidence intervals. (G) Developing cortical plate with immunohistochemical staining for Pax6 (green), Tbr2 (magenta), and tdTomato (red) to characterize progenitors within individual clones at E12.5 following tamoxifen induction at E10.5. Scale bar, 50 µm. (H) High magnification of clone shown in G, demonstrating two PAX6+/Tbr2- radial glia within one proliferative unit. Scale bar, 5 µm. In panels (G) and (H), the images are oriented with the ventricular zone on the left and the pial surface on the right. (I and J) Summary of the number of radial glia and the number of proliferative units per clone for all clones analyzed at E12.5 (n = 30 clones from three mice). Shades of gray in panels (I and J) denote data obtained from different animals for each treatment condition. See also Figure 1—figure supplements 1 and 2 and Figure 1—source datas 1 and 2.
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Figure 1—source data 1
Clone quantification data, related to Figure 1.
Number of cells and clone width for each clone analyzed at postnatal day 10 (P10).
- https://cdn.elifesciences.org/articles/52951/elife-52951-fig1-data1-v3.xlsx
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Figure 1—source data 2
Number and type of progenitors per clone, related to Figure 1.
Number of radial glia, intermediate progenitors and proliferative units for each clone analyzed at embryonic day 12.5 (E12.5).
- https://cdn.elifesciences.org/articles/52951/elife-52951-fig1-data2-v3.xlsx
Figure 1—figure supplement 1
Additional examples and quantitative analyses of clones induced at E11.5 that are missing deep cortical layers, expanding on Figure 1C,F.
(A) Additional examples of clones induced at E11.5 that were lacking neurons in the deep cortical layers. (B) Examples of clones induced at E11.5 that were considered translaminar due to inclusion of neurons in both deep and superficial cortical layers. (C and D) Clones lacking deep-layer neurons (incomplete clones) have fewer neurons overall (C) but there is no difference in clone width between superficial layer-restricted and translaminar clones (D; n = 40 and 10 for translaminar and superficial layer-restricted clones; p-values computed using Wilcoxon rank sum test). Scale bars: 100 µm (A and B).
Figure 1—figure supplement 2
Progenitor composition of clones examined at E12.5 following tamoxifen administration at E10.5, additional examples expanding on Figure 1G–J.
Immunohistochemical staining at E12.5 for Pax6 (green), Tbr2 (magenta), and tdTomato (red) was used to characterize the progenitor composition of clones following tamoxifen administration at E10.5. Examples of clones with 1 (A), 3 (B) or 4 (C) radial glia arranged in 1 (A), 2 (B) and 1 (C) proliferative unit(s) are shown. All panels are oriented with the ventricular zone on the right side and the pial surface on the left. For panels A and B, scale bars are 50 µm on the left and 5 µm on the right, respectively. For panel C, scale bars are 10 µm on the left and 5 µm on the right, respectively. An example with two radial glia arranged in one proliferative unit (the most common pattern) is shown in Figure 1G–H.
Figure 2 with 2 supplements
Region-specific differences in gene expression are present in juvenile mouse neocortex.
(A) Overview of experimental approach using Patch-seq. (B) Example tdTomato-positive translaminar clone spanning cortical layers 2–6 in an acute cortical slice used for Patch-seq experiments. Overlay of bright field and fluorescence image was performed in Adobe Photoshop. Scale bar: 100 μm. (C and D) Box plots showing library size (C) and number of genes detected (D) for all cells passing quality control criteria (n = 206). (E) Density plot of the percent of variance in normalized log-expression values explained by different experimental factors. Each curve corresponds to the variance in gene expression across all genes (n = 12,841 genes) that can be explained by a single variable, with right-shifted curves reflecting variables that explain a higher fraction of the variance. (F) T-distributed stochastic neighbor embedding (t-SNE) plots using the top highly variable and correlated genes across all cells (n = 91 genes; n = 87, 22, 84, and 13 cells in layers 2/3, 4, 5, and 6, respectively), colored by layer position. (G) Performance of a generalized linear model (GLM) trained to predict region from gene expression data of L2/3 neurons (n = 12,841 genes and 85 cells) with model performance (black dot) compared to the chance-level performance estimated using shuffled data (gray, mean and 95% coverage interval; one-tailed p-value computed from shuffled data, shuffling region). (H) Performance of a GLM trained to predict region from gene expression data of L5 neurons (n = 12,841 genes and 77 cells) as described in (G). See also Figure 2—figure supplements 1 and 2 and Figure 2—source data 1.
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Figure 2—source data 1
Gene expression data, related to Figure 2.
Normalized counts, normalized log counts, and metadata for all Patch-seq neurons included in our analysis.
- https://cdn.elifesciences.org/articles/52951/elife-52951-fig2-data1-v3.xls
Figure 2—figure supplement 1
Quality control criteria for single-cell RNA-sequencing data, related to Figure 2C,D.
(A and B) Histograms of library size (A) and number of genes expressed (B) for all sequenced cells. Cells falling more than three median absolute differences below the median (dotted lines) were excluded (n = 11 based on library size and n = 8 based on number of genes expressed; all 8 cells excluded based on number of genes are also excluded based on library size, leaving 206 out of 217 sequenced cells passing these combined criteria). (C) Histogram of average number of counts per cell across genes. Genes with less than one count per cell on average (dotted line) were excluded from further analyses (n = 12,841 genes passing this criteria). (D) Correlation between average number of counts per cell and total number of cells expressing each gene, across genes. (E–G) Library sizes of cells in different layers (E; n = 87, 22, 84, and 13 cells in layers 2/3, 4, 5, and 6 respectively; One-way analysis of variance with post-hoc pairwise comparisons using Tukey’s honestly significant difference procedure), different cortical regions (F; n = 79, 10, and 117 cells in primary somatosensory (SS1), unknown, and primary visual (V1) areas respectively; One-way analysis of variance with post-hoc pairwise comparisons using Tukey’s honestly significant difference procedure), and with (positive) or without (negative) tdTomato-expression (G; n = 110 and 96 negative and positive cells respectively; One-way analysis of variance). (H) Correlation between size factors used for normalization and library size, across cells. (I–K) Number of genes expressed by cells in different layers (I; n = 87, 22, 84, and 13 cells in layers 2/3, 4, 5, and 6 respectively; One-way analysis of variance), in different cortical regions (J; n = 79, 10, and 117 cells in SS1, unknown, and V1 areas respectively; One-way analysis of variance with post-hoc pairwise comparisons using Tukey’s honestly significant difference procedure), and with (positive) or without (negative) tdTomato-expression (K; n = 110 and 96 negative and positive cells respectively; One-way analysis of variance). Values are raw data points expressed as scatter plots (D and H, D with smoothing), binned (A–C), or with overlay violin plots (E–G and I–K). Only significant (p<0.05) p-values are shown (*p<0.05; **p<0.01; ***p<0.001).
Figure 2—figure supplement 2
Expression of top highly variable genes, related to Figure 2F–H.
(A) Variance of normalized log-transformed expression for each gene plotted against the mean log-transformed expression. (B) Violin plots of log-transformed expression values for the top twenty highly variable genes across all cells. (C) T-distributed stochastic neighbor embedding (t-SNE) was performed using the top highly variable and correlated genes (n = 91). Plots are colored by log-transformed expression values for the top twenty highly variable genes.
Figure 3 with 2 supplements
Translaminar clones labeled at E10.5 are composed of diverse transcriptomic subtypes of excitatory neurons.
(A) T-distributed stochastic neighbor embedding (t-SNE) plot showing alignment of our Patch-seq data (data points with black outline, n = 87, 22, 84, and 13 cells in layers 2/3, 4, 5, and 6 respectively) with a recently published mouse cell type atlas (data points with no outline; n = 23,822; from Tasic et al. (2018); colors denote transcriptomic types and are taken from the original publication). The t-SNE of the reference dataset and the positioning of Patch-seq cells were performed as described in Kobak and Berens (2019), see Methods. The size of the Patch-seq data points denotes the precision of the mapping (see Materials and methods): small points indicate high uncertainty. (B) Fraction of labeled (n = 96) and unlabeled (n = 110) cells mapping to specific transcriptomic cell types (cell types with less than three neurons mapped are not shown; overall p=0.039, Chi-squared test). (C and D) Probability of related and unrelated cell pairs mapping to the same transcriptomic cell type either overall (C; n = 337 related pairs, n = 409 unrelated pairs, p=0.70, Chi-squared test) or conditioned on layer position (D; n = 154 related pairs, n = 157 unrelated pairs, p=0.68, Chi-squared test). For (B–D), error bars are 95% Clopper-Pearson confidence intervals and p-values are computed using Chi-squared test (in B, we used Bonferroni correction for each of the 13 post-hoc comparisons). See also Figure 3—figure supplements 1 and 2 and Figure 3—source data 1.
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Figure 3—source data 1
Mapping to transcriptomic cell types, related to Figure 3.
Best match for each of our cells onto reference transcriptomic cell types, t-SNE coordinates for the reference dataset, and t-SNE coordinates for projection of our data onto the reference with a measure of uncertainty.
- https://cdn.elifesciences.org/articles/52951/elife-52951-fig3-data1-v3.xls
Figure 3—figure supplement 1
Translaminar clones labeled at E10.5 are composed of diverse classes of excitatory neurons, related to Figure 3.
(A) T-distributed stochastic neighbor embedding (t-SNE) plot similar to 3A, but with Patch-seq data points (black outline, n = 96 and 110 labeled and unlabeled cells respectively) colored according to tdTomato expression status rather than cortical layer, aligned with a recently published mouse cell type atlas (data points with no outline; n = 23,822; from Tasic et al., 2018; colors denote transcriptomic types and are taken from the original publication). The t-SNE of the reference dataset and the positioning of Patch-seq cells were performed as described in Kobak and Berens (2019), see Materials and methods. The size of the Patch-seq data points denotes the precision of the mapping (see Methods): small points indicate high uncertainty. (B–D) A similar analysis to 3B–D, but using broad cell classes (outlined in panel A) rather than specific transcriptomic cell types. (B) Fraction of labeled (n = 96) and unlabeled (n = 110) cells that mapped to each of the broad classes with greater than three Patch-seq cells total (p=0.38, Chi-squared test). (C and D) Probability of related and unrelated cell pairs mapping to the same broad class either overall (C; n = 337 related pairs, n = 409 unrelated pairs; p=0.71, Chi-squared test) or conditioned on layer position (D; n = 154 related pairs, n = 157 unrelated pairs; p=0.76, Chi-squared test).
Figure 3—figure supplement 2
Transcriptomic diversity of individual translaminar clones, related to Figure 3.
t-SNE plots for each Patch-seq experiment (n = 16) showing clonally related cells in red and unrelated cells in black, projected onto the reference atlas in gray, from Tasic et al. (2018).
Figure 4
Vertical, across-layer connections are selectively increased between excitatory neurons in translaminar clones.
(A–C) Example recording session from four clonally related cells (red) and four nearby, unrelated control cells (black). (A) Morphological reconstruction of all eight neurons. Scale bar, 100 μm. (B) Schematic of connections identified, as well as fluorescence images of each patched cell confirming the overlap of red (lineage tracer) and green (pipette solution) in related cells and green only in control cells. Triangles, pyramidal neurons; ovals, L4 excitatory neurons. (C) Presynaptic action potential (AP) and postsynaptic uEPSC traces for each connection (average of at least 30 trials each). Gray bar indicates period of depolarizing current injection to presynaptic neuron. (D) Connection probabilities among related and unrelated neurons, pooling all connections tested (n = 42/712 potential connections and 1/324 pairs with both directions tested for related neurons; n = 70/1337 potential connections and 6/617 pairs with both directions tested for unrelated neurons). (E) Connection probabilities among related and unrelated neurons, pooling all vertical, across-layer connections tested (n = 28/464 potential connections and 1/211 pairs with both directions tested for related neurons; n = 19/711 potential connections and 0/333 pairs with both directions tested for unrelated neurons). (F) Connection probabilities among related and unrelated neurons, for each vertical connection type tested (n = 0/98, 12/91, 2/75, 6/76, 7/62, and 1/62 potential connections for related neurons and n = 0/141, 12/149, 3/118, 2/123, 2/89, and 0/91 potential connections for unrelated neurons from L2/3 to L4, L4 to L2/3, L5 to L2/3, L2/3 to L5, L4 to L5, and L5 to L4, respectively). (G) Estimated fraction of vertical, across layer input to L2/3 cells (top panel) and L5 cells (bottom panel) coming from clonally related neurons based on our empirically measured clone sizes and connection probabilities. For comparison, the prediction based on previous work (Yu et al., 2009) is shown in black dashed lines. For (D–F), error bars are 95% Clopper-Pearson confidence intervals and p-values are computed using Fisher’s exact test. For (G), error bars and gray dashed lines are propagated standard error of the estimates (see Methods). See also Figure 4—Source data 1.
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Figure 4—source data 1
Summary of connectivity data, related to Figures 4 and 5 and Table 1.
- https://cdn.elifesciences.org/articles/52951/elife-52951-fig4-data1-v3.xlsx
Figure 5 with 3 supplements
Lateral, within-layer connections are not increased between excitatory neurons in translaminar clones.
(A and B) Example recording sessions testing within-layer connections among clonally related cells (red) and nearby, unrelated control cells (black) within L2/3 (A) and L5 (B). Scale bars, 100 μm. Triangles, pyramidal neurons; ovals, L4 excitatory neurons. Presynaptic action potential (AP) and postsynaptic uEPSC traces for each connection are an average of at least 20 trials each. Gray bar indicates period of depolarizing current injection to presynaptic neuron. (C) Connection probabilities among related and unrelated neurons, pooling all lateral, within-layer connections tested (n = 14/248 potential connections and 0/113 pairs with both directions tested for related neurons; n = 51/626 potential connections and 6/284 pairs with both directions tested for unrelated neurons). (D) Connection probabilities among related and unrelated neurons, for each lateral connection type tested (n = 2/105, 11/100, and 1/43 potential connections for related neurons and n = 20/342, 17/148, and 14/136 potential connections for unrelated neurons within L2/3, L4, and L5, respectively). (E) Estimated fraction of lateral inputs to a single cell within L2/3, L4, or L5 that comes from clonally related neurons based on our empirically measured clone sizes and connection probabilities. For comparison, the prediction based on previous work (Yu et al., 2009) is shown in black dashed lines. (F) Heatmap of the log ratio of the connection probabilities for related and unrelated neurons, with additive smoothing (α=1, see Methods) by connection type tested. For (C–D), error bars are 95% Clopper-Pearson confidence intervals and p-values are computed using Fisher’s exact test. For (E), error bars and gray dashed lines are propagated standard error of the estimates (see Methods) For (F), the 95% confidence interval, in parentheses below the value, is computed by resampling; significant values are highlighted in bold. See also Figure 5—figure supplements 1, 2 and 3.
Figure 5—figure supplement 1
Power analysis, related to Figure 5.
Predicted lateral connection probability for related neurons (Prl, panel A) or statistical power of our experiment (Power, panel B) as a function of the estimated number of radial glial lineages pooled in a single clone (N) and the true fold change in connectivity for cells with a shared radial glial lineage (RGC-related, see Methods). Each curve corresponds to a different value of N, as labeled. Red dots denote the values of Prl (A) or Power (B) that would be expected based on the observed increase in vertical connections between clonally related neurons. Blue dashed lines show the actual Prl (A) and fold change (B) that we observed.
Figure 5—figure supplement 2
Comparison of connectivity between clonally related neurons and distance-matched controls.
(A) Connectivity for all connections (left panel); only lateral, within-layer connections (middle panel); or only vertical, across-layer connections (right panel). (B) Connectivity for each layer-defined connection type tested. Pre- and post-synaptic location of the cell bodies is designated above each plot. In A and B, error bars are 95% coverage intervals computed by resampling (see Methods); p-values are two-sided and computed by resampling (see Methods). Related to Figure 5.
Figure 5—figure supplement 3
Connectivity differences between clonally related and unrelated neurons in different rostrocaudal positions.
(A) Connection probabilities for all connection types tested, grouped by rostrocaudal position (n = 449 and 260 related pairs and n = 833 and 485 unrelated pairs in rostral and caudal groups, respectively). (B) Connection probabilities for all vertical, across-layer connections tested, grouped by rostrocaudal position (n = 300 and 163 related pairs and n = 454 and 252 unrelated pairs in rostral and caudal groups, respectively). (C) Connection probabilities for all lateral, within-layer connections tested, grouped by rostrocaudal position (n = 149 and 97 related pairs and n = 379 and 233 unrelated pairs in rostral and caudal groups, respectively). Error bars are 95% Clopper-Pearson confidence intervals and p-values are computed using Fisher’s exact test. Related to Figure 5.
Tables
Table 1
Generalized linear model of connectivity.
Connectivity was modeled as a binomial response variable with the following predictors: lineage relationship (1 for related, 0 for unrelated), connection type (1 for vertical, 0 for lateral), Euclidean distance between the cells in microns, and rostrocaudal position (a numeric factor from 1 to 5; see Materials and methods). ‘×’ denotes an interaction between two linear terms. Overall χ2 = 33.5 compared to constant model, p=2.26 × 10−4, 1988 error degrees of freedom. The four terms with small p-values are: connection class (connection probability P is lower for unrelated vertical connections, compared to unrelated lateral), Euclidean distance (P decreases with increasing distance for unrelated lateral connections), lineage × connection type (P is higher for related vertical pairs), and connection type × Euclidean distance (the effect of Euclidean distance on P depends on the type of connection tested).
Term | Estimated coefficient | SE | t-statistic | p-value |
|---|---|---|---|---|
| Constant | −1.82 | 0.43 | −4.22 | 2.37·10−5 |
| Lineage | −0.47 | 0.58 | −0.81 | 0.42 |
| Connection type | −1.55 | 0.75 | −2.06 | 0.039 |
| Euclidean distance | −8.42·10−3 | 4.40·10−3 | −1.91 | 0.056 |
| Rostrocaudal position | 0.074 | 0.11 | 0.65 | 0.51 |
| Lineage × Connection type | 1.25 | 0.59 | 2.11 | 0.035 |
| Lineage × Euclidean distance | 2.05·10−4 | 2.46·10−3 | 0.083 | 0.93 |
| Lineage × Rostrocaudal position | 0.028 | 0.15 | 0.18 | 0.86 |
| Connection type × Euclidean distance | 7.97·10−3 | 3.99·10−3 | 2.00 | 0.046 |
| Connection type × Rostrocaudal position | 5.86·10−3 | 0.20 | 0.030 | 0.98 |
| Euclidean distance × Rostrocaudal position | −5.97·10−4 | 9.33·10−4 | −0.64 | 0.52 |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene Mus musculus | nestin | MGI:101784 NCBI Gene: 18008 | ||
| Strain, strain background Mus musculus | C57Bl/6J | The Jackson Laboratory | JAX Stock no. 000664 RRID:IMSR_JAX:000664 | Females |
| Strain, strain background Mus musculus | CD1 | Obtained from the Baylor College of Medicine Center for Comparative Medicine | ||
| Strain, strain background Mus musculus | Nestin-CreER | Obtained from Dr. Mirjana Maletic-Savatic Lab at Baylor College of Medicine | Cryopreserved by Andreas Tolias Lab at Baylor College of Medicine | |
| Strain, strain background Mus musculus | Ai9 | The Jackson Laboratory | JAX Stock no. 007909 RRID:IMSR_JAX:007909 | |
| Antibody | Rabbit anti-Tbr2 | Abcam | cat. no. AB23345 RRID:AB_778267 | |
| Antibody | Mouse anti-Pax6 | Developmental Studies Hybridoma Bank (DSHB) at the University of Iowa. PAX6 was deposited to the DSHB by Kawakami, A. | DSHB Hybridoma Product PAX6 RRID:AB_528427 | |
| Antibody | Goat anti-tdTomato | Sicgen | cat. no. AB8181-200 RRID:AB_2722750 | |
| Chemical compound, drug | Tamoxifen (≥99%) | Sigma-Aldrich | cat. no. T5648 | |
| Chemical compound, drug | Progesterone (≥99%) | Sigma-Aldrich | cat. no. P0130 | |
| Other | Corn Oil | Sigma-Aldrich | cat. no. C8267 | |
| Other | DNA-OFF | Clontech | Cat. no 9036 | |
| Other | RNase Zap | Thermo Fisher Scientific | Cat. no. AM9780 | |
| Peptide, recombinant protein | Recombinant RNase inhibitor | Clontech | 2313A | |
| Other | Potassium D-gluconate (K-gluconate,≥99%) | Sigma-Aldrich | cat. no 64500 | |
| Other | Potassium chloride (KCl, for molecular biology,≥99.0%) | Sigma-Aldrich | cat. no. P9541 | |
| Other | HEPES solution (1 M, BioReagent) | Sigma-Aldrich | cat. no. H3537 | |
| Other | Ethylene glycol-bis(2-aminoethylether)-N,N,N’,N’-tetraacetic acid (EGTA, for molecular biology,≥97.0%) | Sigma-Aldrich | cat. no. E3889 | |
| Other | Adenosine 5’-triphosphate magnesium salt (Mg-ATP,≥95%) | Sigma-Aldrich | cat. no. A9187 | |
| Other | Guanosine 5’-triphosphate sodium salt hydrate (Na-GTP,≥90%) | Sigma-Aldrich | cat. no. 51120 | |
| Other | Phosphocreatine disodium salt hydrate (Na2-phosphocreatine,≥97%) | Sigma-Aldrich | cat. no. P7936 | |
| Other | Glycogen (RNA grade) | Thermo Fisher Scientific | cat. no. R0551 | |
| Other | Biocytin (≥98%) | Sigma-Aldrich | cat. no. B4261 | |
| Sequence-based reagent | ERCC RNA spike-in mix | Thermo Fisher Scientific | cat. no. 4456740 | sequences available at website |
| Other | Tris-EDTA buffer solution (TE buffer, BioUltra, for molecular biology) | Sigma-Aldrich | cat. no. 93283 | |
| Other | Triton X-100 | Sigma-Aldrich | cat. no. T8787 | |
| Other | Betaine (BioUltra,≥99.0%) | Sigma-Aldrich | cat. no. 61962 | |
| Other | dNTPs (25 mM each) | Thermo Fisher Scientific | cat. no. R1121 | |
| Peptide, recombinant protein | Superscript II reverse transcriptase (SSIIRT) | Thermo Fisher Scientific | cat. no. 18064014 | |
| Other | MgCl2 (1M, molecular biology grade) | Thermo Fisher Scientific | cat. no. AM9530G | |
| Commercial assay, kit | KAPA Biosystems HiFi HotStart Ready Mix | Thermo Fisher Scientific | cat. no. NO0295239 | |
| Other | TAPS (≥99.5%) | Sigma-Aldrich | cat. no. T5130 | |
| Other | Polyethylene glycol solution (PEG-8000, 40% wt/vol) | Sigma-Aldrich | cat. no. P1458 | |
| Commercial assay, kit | Nextera XT index kit v2 set A for 96 indices, 384 samples | Illumina | cat. no. FC-131–2001 | |
| Commercial assay, kit | Axygen AxyPrep mag PCR clean-up kit | Thermo Fisher Scientific | cat. no. 14223151 | |
| Commercial assay, kit | KAPA HiFi PCR Kit | KAPA Biosystems | cat. no. KK2103 | |
| Other | Hoechst 33342 | Invitrogen | cat. no. H3570 | |
| Other | Antigen unmasking solution | Vector Laboratories | cat. no. H-3300 | |
| Other | Vectashield antifade mounting medium | Vector Laboratories | cat. no. H-1000 | |
| Sequence-based reagent | Oligo-dT30VN (HPLC purified) | Biomers.net | oligonucleotide | 5’- AAGCAGTGGTATCAACGCAGAGTACTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN-3’, where V represents A, C or G and N represents any nucleotide |
| Sequence-based reagent | IS PCR Oligo (HPLC purified) | Biomers.net | oligonucleotide | 5’-AAGCAGTGGTATCAACGCAGAGT-3’ |
| Sequence-based reagent | Template switching oligonucleotide (LNA-TSO; RNase-free; HPLC purified | Exiqon | oligonucleotide | 5’- AAGCAGTGGTATCAACGCAGAGTACrGrG+G-3’, where rG indicates riboguanosines and +G indicates a locked nucleic acid (LNA)-modified guanosine |
| Software, algorithm | Patchmaster software | HEKA | RRID:SCR_000034 | |
| Software, algorithm | STAR v2.4.2a | https://github.com/alexdobin/STAR/releases/tag/STAR_2.4.2a | RRID:SCR_015899 | |
| Software, algorithm | Neurolucida | MBF Bioscience | RRID:SCR_001775 | |
| Other | Glass capillaries (2.0 mm OD, 1.16 mm ID) | Sutter Instruments |
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Cell type composition and circuit organization of clonally related excitatory neurons in the juvenile mouse neocortex
eLife 9:e52951.
https://doi.org/10.7554/eLife.52951
