Journal: Cell genomics

Article Title: Genetic and functional analysis of Raynaud's syndrome implicates loci in vasculature and immunity.

doi: 10.1016/j.xgen.2024.100630

Figure Lengend Snippet: Figure 1. Meta-analysis (A) A Manhattan plot of RS meta-analysis (11,358 cases and 1,106,871 controls) combining UKB, FinnGen, MGB Biobank, and Estonian Biobank (EstBB) (b38). (B and C) Locus zoom plots for (B) regional association at the ADRA2A locus (b38), lead variant rs7090046, and (C) regional association at the NOS3 locus (b38), lead variant rs3918226.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies ADRA2A probe ACDBio 602791 IRX1 probe ACDBio 1043401 Bacterial and virus strains Lentivirus 2ND generation pCMV-dR8.91 Gilbert et al.68 and Horlbeck et al.69 Addgene 8455 Lentivirus 2ND generation, pMD2.G Trono lab, unpublished Addgene 12259 NEB Stable Competent E. coli, High Efficiency NEB C3040H NEB 5-alpha Competent E. coli (High Efficiency) NEB C2987H Biological samples De-identified human skin biopsy samples This paper Stanford University, Department of Dermatology Critical commercial assays Cyto-Select 48-well Cell Contraction Assay Kit Cell Biolabs N/A PrimeFlowTM RNA Assay Kit ThermoFisher Scientific 88-18005-210 RNAscopeTM 2.5 HD Assay-Red and ACD RNAscope 2.5 HD Duplex Assay ACD Bio N/A RNAiMax Invitrogen, Carlsbad, CA 13778150 Active Motif ATAC-seq kit Active motif 53150 Deposited data Meta-analysis summary statistics This paper Dryad open repository (https://doi.org/10.5061/dryad.1g1jwsv53) FinnGen Kurki et al.70 https://www.finngen.fi/en/access_results UK Biobank Bycroft et al.71 https://www.ukbiobank.ac.uk/enable- your-research/apply-for-access The Estonian Biobank Leitsalu et al.72 releases@ut.ee The Mass General Brigham Biobank Boutin et al.73 https://www.partners.org/Medical- Research/Support-Offices/HumanResearch-Committee-IRB/Default.aspx GTEx eQTL data GTEx Consortium74 https://gtexportal.org/home/ Skin scRNAseq data He et al.75 Reposited in GEO under GSE147424.

Techniques: Variant Assay

Journal: Cell genomics

Article Title: Genetic and functional analysis of Raynaud's syndrome implicates loci in vasculature and immunity.

doi: 10.1016/j.xgen.2024.100630

Figure Lengend Snippet: Figure 2. ADRA2A expression (A and B) Genotype tissue expression for SNP rs7090046 in (A) tibial arteries and (B) across tissues from GTEx (NES, normalized expression values; m-value = posterior probability, p % 0.05). (C) The RS association co-localizes with eQTL signal in tibial arteries. Created with Biorender.com.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies ADRA2A probe ACDBio 602791 IRX1 probe ACDBio 1043401 Bacterial and virus strains Lentivirus 2ND generation pCMV-dR8.91 Gilbert et al.68 and Horlbeck et al.69 Addgene 8455 Lentivirus 2ND generation, pMD2.G Trono lab, unpublished Addgene 12259 NEB Stable Competent E. coli, High Efficiency NEB C3040H NEB 5-alpha Competent E. coli (High Efficiency) NEB C2987H Biological samples De-identified human skin biopsy samples This paper Stanford University, Department of Dermatology Critical commercial assays Cyto-Select 48-well Cell Contraction Assay Kit Cell Biolabs N/A PrimeFlowTM RNA Assay Kit ThermoFisher Scientific 88-18005-210 RNAscopeTM 2.5 HD Assay-Red and ACD RNAscope 2.5 HD Duplex Assay ACD Bio N/A RNAiMax Invitrogen, Carlsbad, CA 13778150 Active Motif ATAC-seq kit Active motif 53150 Deposited data Meta-analysis summary statistics This paper Dryad open repository (https://doi.org/10.5061/dryad.1g1jwsv53) FinnGen Kurki et al.70 https://www.finngen.fi/en/access_results UK Biobank Bycroft et al.71 https://www.ukbiobank.ac.uk/enable- your-research/apply-for-access The Estonian Biobank Leitsalu et al.72 releases@ut.ee The Mass General Brigham Biobank Boutin et al.73 https://www.partners.org/Medical- Research/Support-Offices/HumanResearch-Committee-IRB/Default.aspx GTEx eQTL data GTEx Consortium74 https://gtexportal.org/home/ Skin scRNAseq data He et al.75 Reposited in GEO under GSE147424.

Techniques: Expressing

Journal: Cell genomics

Article Title: Genetic and functional analysis of Raynaud's syndrome implicates loci in vasculature and immunity.

doi: 10.1016/j.xgen.2024.100630

Figure Lengend Snippet: Figure 3. ADRA2A expression is restricted to SMCs located in distal arterioles (A–D) Uniform manifold approximation and projection (UMAP) plot of (A) human skin and (B) human coronary artery scRNA-seq indicating ADRA2A expression, co-expression of NOTCH3 in the same cluster, and MYH11 expression as SMC cluster and all clusters in skin (C) and in the coronary artery dataset (D). (E) A schematic of vascular wall and ADRA2A expression. (F) RNAscope for ADRA2A in a dorsal hand biopsy. (G) RT-PCR quantification of ADRA2A and NOTCH expression in human arterial SMCs and pulmonary arteriolar. Mean ± SEM, ***p < 0.0005, **p < 0.005, and *p < 0.05 (unpaired two-tailed Student’s t test).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies ADRA2A probe ACDBio 602791 IRX1 probe ACDBio 1043401 Bacterial and virus strains Lentivirus 2ND generation pCMV-dR8.91 Gilbert et al.68 and Horlbeck et al.69 Addgene 8455 Lentivirus 2ND generation, pMD2.G Trono lab, unpublished Addgene 12259 NEB Stable Competent E. coli, High Efficiency NEB C3040H NEB 5-alpha Competent E. coli (High Efficiency) NEB C2987H Biological samples De-identified human skin biopsy samples This paper Stanford University, Department of Dermatology Critical commercial assays Cyto-Select 48-well Cell Contraction Assay Kit Cell Biolabs N/A PrimeFlowTM RNA Assay Kit ThermoFisher Scientific 88-18005-210 RNAscopeTM 2.5 HD Assay-Red and ACD RNAscope 2.5 HD Duplex Assay ACD Bio N/A RNAiMax Invitrogen, Carlsbad, CA 13778150 Active Motif ATAC-seq kit Active motif 53150 Deposited data Meta-analysis summary statistics This paper Dryad open repository (https://doi.org/10.5061/dryad.1g1jwsv53) FinnGen Kurki et al.70 https://www.finngen.fi/en/access_results UK Biobank Bycroft et al.71 https://www.ukbiobank.ac.uk/enable- your-research/apply-for-access The Estonian Biobank Leitsalu et al.72 releases@ut.ee The Mass General Brigham Biobank Boutin et al.73 https://www.partners.org/Medical- Research/Support-Offices/HumanResearch-Committee-IRB/Default.aspx GTEx eQTL data GTEx Consortium74 https://gtexportal.org/home/ Skin scRNAseq data He et al.75 Reposited in GEO under GSE147424.

Techniques: Expressing, RNAscope, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test

Journal: Cell genomics

Article Title: Genetic and functional analysis of Raynaud's syndrome implicates loci in vasculature and immunity.

doi: 10.1016/j.xgen.2024.100630

Figure Lengend Snippet: Figure 4. CRISPRi against rs7090046 supports causal role of ADRA2A (A) Schematic of the CRISPRi experiment. (B) RT-PCR quantification of ADRA2A expression in rs7090046 guide targeted cells vs. CTRL. Mean ± SEM, ***p < 0.0005, **p < 0.005, and *p < 0.05 (one-way ANOVA).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies ADRA2A probe ACDBio 602791 IRX1 probe ACDBio 1043401 Bacterial and virus strains Lentivirus 2ND generation pCMV-dR8.91 Gilbert et al.68 and Horlbeck et al.69 Addgene 8455 Lentivirus 2ND generation, pMD2.G Trono lab, unpublished Addgene 12259 NEB Stable Competent E. coli, High Efficiency NEB C3040H NEB 5-alpha Competent E. coli (High Efficiency) NEB C2987H Biological samples De-identified human skin biopsy samples This paper Stanford University, Department of Dermatology Critical commercial assays Cyto-Select 48-well Cell Contraction Assay Kit Cell Biolabs N/A PrimeFlowTM RNA Assay Kit ThermoFisher Scientific 88-18005-210 RNAscopeTM 2.5 HD Assay-Red and ACD RNAscope 2.5 HD Duplex Assay ACD Bio N/A RNAiMax Invitrogen, Carlsbad, CA 13778150 Active Motif ATAC-seq kit Active motif 53150 Deposited data Meta-analysis summary statistics This paper Dryad open repository (https://doi.org/10.5061/dryad.1g1jwsv53) FinnGen Kurki et al.70 https://www.finngen.fi/en/access_results UK Biobank Bycroft et al.71 https://www.ukbiobank.ac.uk/enable- your-research/apply-for-access The Estonian Biobank Leitsalu et al.72 releases@ut.ee The Mass General Brigham Biobank Boutin et al.73 https://www.partners.org/Medical- Research/Support-Offices/HumanResearch-Committee-IRB/Default.aspx GTEx eQTL data GTEx Consortium74 https://gtexportal.org/home/ Skin scRNAseq data He et al.75 Reposited in GEO under GSE147424.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: Cell genomics

Article Title: Genetic and functional analysis of Raynaud's syndrome implicates loci in vasculature and immunity.

doi: 10.1016/j.xgen.2024.100630

Figure Lengend Snippet: Figure 5. ADRA2A expression affects SMC contraction upon cold stimulus (+28C) (A) ADRA2A and ADRA2C silencing in cold exposure. (B) ADRA2A and ADRA2C silencing in ambient conditions. (C) ADRA2A and ADRA2C overexpression in cold exposure. (D) ADRA2A and ADRA2C overexpression in ambient conditions. Mean ± SEM, ***p < 0.0005, **p < 0.005, and *p < 0.05 (unpaired two-tailed Student’s t test).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies ADRA2A probe ACDBio 602791 IRX1 probe ACDBio 1043401 Bacterial and virus strains Lentivirus 2ND generation pCMV-dR8.91 Gilbert et al.68 and Horlbeck et al.69 Addgene 8455 Lentivirus 2ND generation, pMD2.G Trono lab, unpublished Addgene 12259 NEB Stable Competent E. coli, High Efficiency NEB C3040H NEB 5-alpha Competent E. coli (High Efficiency) NEB C2987H Biological samples De-identified human skin biopsy samples This paper Stanford University, Department of Dermatology Critical commercial assays Cyto-Select 48-well Cell Contraction Assay Kit Cell Biolabs N/A PrimeFlowTM RNA Assay Kit ThermoFisher Scientific 88-18005-210 RNAscopeTM 2.5 HD Assay-Red and ACD RNAscope 2.5 HD Duplex Assay ACD Bio N/A RNAiMax Invitrogen, Carlsbad, CA 13778150 Active Motif ATAC-seq kit Active motif 53150 Deposited data Meta-analysis summary statistics This paper Dryad open repository (https://doi.org/10.5061/dryad.1g1jwsv53) FinnGen Kurki et al.70 https://www.finngen.fi/en/access_results UK Biobank Bycroft et al.71 https://www.ukbiobank.ac.uk/enable- your-research/apply-for-access The Estonian Biobank Leitsalu et al.72 releases@ut.ee The Mass General Brigham Biobank Boutin et al.73 https://www.partners.org/Medical- Research/Support-Offices/HumanResearch-Committee-IRB/Default.aspx GTEx eQTL data GTEx Consortium74 https://gtexportal.org/home/ Skin scRNAseq data He et al.75 Reposited in GEO under GSE147424.

Techniques: Expressing, Over Expression, Two Tailed Test

Journal: Cell genomics

Article Title: Genetic and functional analysis of Raynaud's syndrome implicates loci in vasculature and immunity.

doi: 10.1016/j.xgen.2024.100630

Figure Lengend Snippet: Figure 6. Schematic of the proposed RS-associated pathomechanism We propose a possible mechanism based on our observations from genetic and contraction assays. In this hypothesized model, the pathomechanism of RS is dependent on ADRA2A expression. In healthy patients without RS, there are mechanisms to prevent unwanted and excessive vessel contraction. First, the cell constriction (upon cold or stress) can be limited by the increased release of ligand by translocation of the a2C-adrenergic receptor from the cytosol to the cell surface. Second, a2A-adrenergic receptors are also expressed on the presynaptic membrane and function there as a negative feedback loop for catecholamine release. In patients with RS, the expression of the ADRA2A gene, that encodes the a2A-adrenergic receptor, is higher, and therefore the number of a2A-adrenergic receptor available for ligand binding on microvascular SMCs is also higher. Increase in SMC ADRA2A expression sensitizes the postsynaptic system that ag- gravates the adrenergic effects of SMC contraction. In conditions such as cold and stress where more ligand is released, the contraction is further accentuated. Black arrows represent adrenergic downstream signaling strength.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies ADRA2A probe ACDBio 602791 IRX1 probe ACDBio 1043401 Bacterial and virus strains Lentivirus 2ND generation pCMV-dR8.91 Gilbert et al.68 and Horlbeck et al.69 Addgene 8455 Lentivirus 2ND generation, pMD2.G Trono lab, unpublished Addgene 12259 NEB Stable Competent E. coli, High Efficiency NEB C3040H NEB 5-alpha Competent E. coli (High Efficiency) NEB C2987H Biological samples De-identified human skin biopsy samples This paper Stanford University, Department of Dermatology Critical commercial assays Cyto-Select 48-well Cell Contraction Assay Kit Cell Biolabs N/A PrimeFlowTM RNA Assay Kit ThermoFisher Scientific 88-18005-210 RNAscopeTM 2.5 HD Assay-Red and ACD RNAscope 2.5 HD Duplex Assay ACD Bio N/A RNAiMax Invitrogen, Carlsbad, CA 13778150 Active Motif ATAC-seq kit Active motif 53150 Deposited data Meta-analysis summary statistics This paper Dryad open repository (https://doi.org/10.5061/dryad.1g1jwsv53) FinnGen Kurki et al.70 https://www.finngen.fi/en/access_results UK Biobank Bycroft et al.71 https://www.ukbiobank.ac.uk/enable- your-research/apply-for-access The Estonian Biobank Leitsalu et al.72 releases@ut.ee The Mass General Brigham Biobank Boutin et al.73 https://www.partners.org/Medical- Research/Support-Offices/HumanResearch-Committee-IRB/Default.aspx GTEx eQTL data GTEx Consortium74 https://gtexportal.org/home/ Skin scRNAseq data He et al.75 Reposited in GEO under GSE147424.

Techniques: Expressing, Translocation Assay, Membrane, Ligand Binding Assay

Journal: Nature

Article Title: Distinct subnetworks of the thalamic reticular nucleus

doi: 10.1038/s41586-020-2504-5

Figure Lengend Snippet: (a) Illustration of TRN gating on corticothalamo (CT) circuitry and experiments. Green arrows illustrate CT projection passing through TRN. MO: motor cortex; SS: somatosensory cortex; Aud: auditory cortex; Vis: visual cortex. Structure in red: TRN tissue ( TdTomato ). (b) t-SNE embedding shows nucleus clusters, indicated by colors. GABAergic cells (GABA), oligodendrocytes (ODC), Glutamatergic cells (Glut), oligodendrocyte precursor cells (OPC), microglia (MCG), astrocytes (ASC). n = 1,687 nuclei. (c) biSNE embedding of Gad2 + and Pvalb + single nuclei, pseudo-colored by gradient score (GS), (d) by Spp1 (left) or Ecel1 (right) expression, showing two extremes marked by Spp1 or Ecel1 , respectively. n = 671 nuclei. (e) Heat map showing Spp1- and Ecel1- associated transcriptional programs. Some genes relevant to electrophysiological properties are highlighted on the side. Columns: single nuclei; Rows: genes. (f) Scatter plot showing the percentage of ‘Spp1’ and ‘Ecel1’ profile-specific genes expressed in individual TRN cells along the transcriptomic gradient. (g) Distribution of TRN Spp1 + , Ecel1 + and intermediate populations DN and DP, along the transcriptomic gradient.

Article Snippet: RNAscope probes used were Mm-Spp1-C1 (ACDBio, USA, #435191, NM_001204201.1, region 2-1079), Mm-Ecel1-C3 (ACDBio, USA, #475331-C3, NM_001277925.1, region 879-1992), and Mm-Pvalb-C4 (ACDBio, USA, #421931-C4, NM_013645.3, region 2*885).

Techniques: Expressing

Journal: Nature

Article Title: Distinct subnetworks of the thalamic reticular nucleus

doi: 10.1038/s41586-020-2504-5

Figure Lengend Snippet: (a) Expression levels of Spp1 and Ecel1 (upper) and log( Spp1 / Ecel1 ) (lower) in individual cells along the transcriptomic gradient, showing binary pattern of Spp1 and Ecel1 correlated with the gradient score. (b) Number of genes detected for the major cell types and TRN subpopulations, showing that the DP and DN are of quality comparable to other cell populations. n ASC =124, n Glut =226, n GABA =868, n ODC =388, n Gad1 + Cck + =9, n OPC =23, n MCG =28, n Ebf2 + =21 nuclei. Between Spp1 + and DP, p=0.1787; DN and Ecel1 + , p=0.2897, two-sided ranksum test. n.s.: not significant. No adjustment for multiple testing was applicable. Box plots shows 25th, 50th, 75th percentiles, and the whiskers extend to the most extreme data points, ‘+’ are taken as outliers (Matlab R2017a). (c) Schematics of naïve Bayes classifiers to assign Spp1 + , Ecel1 + , DP and DN neurons into segments of the transcriptomic gradient. (d) Classification accuracy of the naïve Bayes classifiers. Shown are the probability of assigning Spp1 + to ‘Spp1’ segment, DP & DN neuron to the intermediate segment, and Ecel1 + to ‘Ecel1’ segment respectively. n=671 nuclei total, n Spp1 + =264, n DP+DN =195, n Ecel1 + =212 neurons. (e) Schematics for normalization of medial-lateral position of individual neurons in FISH images. Blue line: TRN boundary; Red dots: Pvalb + Spp1 + neurons; Green dots: Pvalb + Ecel1 + neurons; Yellow dots: DP neurons; DN are not shown in the schematics. (f) Scatter plots showing log( Spp1 / Ecel1 ) in individual Pvalb + neurons at normalized medial-lateral position in selected tFISH images along anterior to posterior TRN, corresponding to . m-l: medial-lateral position; Blue dots: individual cells; Solid red line: smooth fitting of blue data points, showing inverted-’U’ shape; Dashed blue line: mean of blue data points, indicating the difference in the FISH background of Spp1 and Ecel1 channel in different images.

Article Snippet: RNAscope probes used were Mm-Spp1-C1 (ACDBio, USA, #435191, NM_001204201.1, region 2-1079), Mm-Ecel1-C3 (ACDBio, USA, #475331-C3, NM_001277925.1, region 879-1992), and Mm-Pvalb-C4 (ACDBio, USA, #421931-C4, NM_013645.3, region 2*885).

Techniques: Expressing

Journal: Nature

Article Title: Distinct subnetworks of the thalamic reticular nucleus

doi: 10.1038/s41586-020-2504-5

Figure Lengend Snippet: (a) RNA-FISH co-staining in coronal sections. TRN region is delineated based on Pvalb FISH signal. Scale bar: 200μm. D: dorsal; V: ventral; L: lateral; M: medial. IC: internal capsule; AM: anteromedial nucleus; AVVL: anteroventral nucleus; VPL: ventral posterolateral nucleus. Repeated with n = 9. The boxed area in image 5 is zoomed in (b) . arrow: DP neurons; arrow-head, DN neurons. (c) Fraction of TRN Pvalb + cells stained Spp1 + , Ecel1 + , DN or DP, quantified in RNA-FISH co-staining in coronal sections along anterior (A) to posterior (P) axis. Insert: Schematics of TRN coronal sections.

Article Snippet: RNAscope probes used were Mm-Spp1-C1 (ACDBio, USA, #435191, NM_001204201.1, region 2-1079), Mm-Ecel1-C3 (ACDBio, USA, #475331-C3, NM_001277925.1, region 879-1992), and Mm-Pvalb-C4 (ACDBio, USA, #421931-C4, NM_013645.3, region 2*885).

Techniques: Staining

Journal: Nature

Article Title: Distinct subnetworks of the thalamic reticular nucleus

doi: 10.1038/s41586-020-2504-5

Figure Lengend Snippet: (a) Right: Experiment schematics illustrating injection of retrograde fluorescent beads (RetroBeads) in first-order (FO) or higher-order (HO) thalamic nuclei and retrograde tracing to the projecting TRN neurons. Left: an example illustrating tracing from FO (dLGN) and HO (LP) visual thalamic nuclei. (b) Percentage of Spp1 + , Ecel1 + , DP and DN cells labeled by RetroBeads traced from injections targeting different thalamic nuclei, (p=1.45×10 −26 , two-sided χ-square test. Bars represent mean ± SD). (c) Retrograde tracing of TRN neurons by projecting to FO thalamic nuclei (dLGN, VPM and vMGN, left column) and HO nuclei (LP, POm and dMGN, right column) overlapped with RNA-FISH co-staining for Spp1 and Ecel1 , showing high overlap of RetroBeads and Spp1 + staining when FO nuclei were injected and high overlap of RetroBeads and Ecel1 + when HO nuclei were injected. Separate channels are shown in the columns on the right side (upper row panels: injection in FO nuclei; lower row panels: injection in HO nuclei). VL: ventral lateral nucleus; ST: stria terminalis; VPL: ventral posterolateral nucleus. For (b) and (c), n = 3 mice per region.

Article Snippet: RNAscope probes used were Mm-Spp1-C1 (ACDBio, USA, #435191, NM_001204201.1, region 2-1079), Mm-Ecel1-C3 (ACDBio, USA, #475331-C3, NM_001277925.1, region 879-1992), and Mm-Pvalb-C4 (ACDBio, USA, #421931-C4, NM_013645.3, region 2*885).

Techniques: Injection, Retrograde Tracing, Labeling, Staining

Journal: Nature

Article Title: Distinct subnetworks of the thalamic reticular nucleus

doi: 10.1038/s41586-020-2504-5

Figure Lengend Snippet: (a) The positions of retrogradely labeled neurons traced from different thalamic relay nuclei indicated by different colors are shown in the coronal view of TRN section series arranged from anterior to posterior. The light green and the magenta shaded areas indicate the distribution of typical Ecel1 + and Spp1 + neurons, respectively. VPM, ventral posterior medial; dLGN, lateral geniculate nucleus (dorsal part); POm, posterior-medial; LP, lateral posterior-lateral part; dMGN, medial geniculate-dorsal part; vMGN, medial geniculate-ventral part; VM, ventromedial; VL, ventrolateral. n = 3 mice per region. (b) Panoramic view of coronal sections showing the injection sites and cortical projection for each FO and HO thalamic nuclei. V1: primary visual cortex; V2L: secondary visual cortex lateral part; V2ML: secondary visual cortex medial-lateral part; S1BF: primary somatosensory cortex barrel field; S1DZ: primary somatosensory cortex dysgranular zones; S1FL: primary somatosensory cortex forelimb; S1HL: primary somatosensory cortex hindlimb; S2: secondary somatosensory cortex; AuV: secondary auditory cortex ventral area; Au1: primary auditory cortex; AuD: secondary auditory cortex dorsal area; MGD: medial geniculate nuclei dorsal part; MGM: medial geniculate nuclei medial part; TeA: temporal cortex, association area. n = 3 mice per region. (c) Quantification of the projection ratio between the primary and higher-order secondary/tertiary cortical areas for different thalamic injection sites. n dLGN =12 slices/2 mice, n LP =12 slices/2 mice, n MGV =15 slices/2 mice, n MGD/MGM =12 slices/2 mice, n VPM =12 slices/2 mice, n POm =12 slices/2 mice. Bars represent mean ± SEM and raw data points.

Article Snippet: RNAscope probes used were Mm-Spp1-C1 (ACDBio, USA, #435191, NM_001204201.1, region 2-1079), Mm-Ecel1-C3 (ACDBio, USA, #475331-C3, NM_001277925.1, region 879-1992), and Mm-Pvalb-C4 (ACDBio, USA, #421931-C4, NM_013645.3, region 2*885).

Techniques: Labeling, Injection

Journal: Nature

Article Title: Distinct subnetworks of the thalamic reticular nucleus

doi: 10.1038/s41586-020-2504-5

Figure Lengend Snippet: (a) Left: Schematics of experiments. Right: Localization of Patch-Seq neurons in TRN coronal sections. (b) Example of the recording protocol to measure bursting firing: neurons were held at different membrane potentials followed by a hyperpolarizing pulse injection, and traces were quantified for different parameters including the maximum number of rebound bursts. (c) Summary of rebound burst properties (p=0.0001 for all three parameters, two-sided unpaired t-test). Mean ± SD and raw data points. Spp1 + (n=29), Ecel1 + (n=15), DP (n=10) and DN (n=13) neurons collected from 5 mice. (d) Number of rebound bursts generated at different membrane potentials (bars represent mean ± SD). Spp1 + (n=24), Ecel1 + (n=15), DP (n=9) and DN (n=10) neurons collected from 5 mice. (e) The 3D plot of rebound bursts #, frequency within the first rebound burst (rbFreq) and AHP, showing the continuous distribution pattern of Ecel1 + (n=15), DP (n=9),DN (n=10), and Spp1 + (n=29) neurons, collected from 5 mice. (f) Correlation of the gradient score with the maximum burst # (n=59 neurons). Line represents linear regression fitting.

Article Snippet: RNAscope probes used were Mm-Spp1-C1 (ACDBio, USA, #435191, NM_001204201.1, region 2-1079), Mm-Ecel1-C3 (ACDBio, USA, #475331-C3, NM_001277925.1, region 879-1992), and Mm-Pvalb-C4 (ACDBio, USA, #421931-C4, NM_013645.3, region 2*885).

Techniques: Membrane, Injection, Generated

Journal: Nature

Article Title: Distinct subnetworks of the thalamic reticular nucleus

doi: 10.1038/s41586-020-2504-5

Figure Lengend Snippet: (a) The anatomical distribution of Patch-seq recorded neurons in coronal sections of TRN along the anterior-posterior axis. The cells were labeled with different colored numbers as indicated ( Spp1 + , magenta, Ecel1 + , green, DP, black and DN, blue). Numbers indicate cell id. Shown are n=76 cells/5 mice, data collected by 2 experimenters. (b) biSNE embedding of the collected TRN neurons for Patch-seq showing molecular gradient pattern with Spp 1 + (magenta), Ecel 1 + (green), and the intermediate sub-populations DP (blue) and DN (black). Shown are subset of neurons from a batch of n=68 neurons/5 mice. (c) Representative voltage changes in response to hyperpolarizing current step injections. Spp1 + neurons (magenta) show robust rebound burst firings elicited by hyperpolarization with high firing frequencies within a burst. When a similar protocol is applied, most of the Ecel1 + neurons (green) show only one rebound burst with lower firing frequencies within a burst than Spp1 + neurons. DN (blue) and DP (black) neurons present intermediate properties. n Ecel1 + = 15, n Spp1 + = 29, n DN = 9, n DP = 10 neurons.

Article Snippet: RNAscope probes used were Mm-Spp1-C1 (ACDBio, USA, #435191, NM_001204201.1, region 2-1079), Mm-Ecel1-C3 (ACDBio, USA, #475331-C3, NM_001277925.1, region 879-1992), and Mm-Pvalb-C4 (ACDBio, USA, #421931-C4, NM_013645.3, region 2*885).

Techniques: Labeling

Journal: Nature

Article Title: Distinct subnetworks of the thalamic reticular nucleus

doi: 10.1038/s41586-020-2504-5

Figure Lengend Snippet: (a) Zoom-in view of a representative single action potential traces of Spp 1 + and Ecel1 + neurons. (b) Summary of action potential (AP) threshold (p=0.015, two-sided unpaired t-test) and half-width of AP (APhw) (p= 7.08×10 5 , two-sided unpaired t-test). For (a) and (b), n Spp1 =12, n Ecel1 =13, n DP =6, n DN =7 neurons from 5 mice. Plots represent mean ± SD and raw data points. (c) Example of Spp1 + like (‘Spp1’) (magenta) and Ecel1 + like (‘Ecel1’) (green) neuron morphology. (d) Sholl analysis of the dendritic complexity. (e) Summary of the soma length and width, total dendritic length and maximum number of intersections in Spp1 + like (‘Spp1’) and Ecel1 + like (‘Ecel1’) neurons (Mean ± SD. Dendritic length, p=0.0014. Number of intersections, p=0.0004, two-sided unpaired t-test). For (c), (d), and (e), n ’Spp1’ =11 neurons/4 mice, n ’Ecel1’ =10 neurons/4 mice.

Article Snippet: RNAscope probes used were Mm-Spp1-C1 (ACDBio, USA, #435191, NM_001204201.1, region 2-1079), Mm-Ecel1-C3 (ACDBio, USA, #475331-C3, NM_001277925.1, region 879-1992), and Mm-Pvalb-C4 (ACDBio, USA, #421931-C4, NM_013645.3, region 2*885).

Techniques:

Journal: Nature

Article Title: Distinct subnetworks of the thalamic reticular nucleus

doi: 10.1038/s41586-020-2504-5

Figure Lengend Snippet: (a) Schematics of the AAV-mediated pooled CRISPR/Cas9 in vivo screen. (b) List of pools and genes selected for knockout in the CRISPR/ Cas9 screening. TRN enriched refers to genes differentially expressed between Pvalb + neurons from TRN and from M2 cortex, somatosensory cortex, striatum, and hippocampus. (c) A heat map showing the expression pattern of the selected genes in the TRN neurons. The selected disease-risk genes are labeled on the right side. (d) A heat map showing the differentially expressed disease-risk genes in Spp1 + versus Ecel1 + neurons: autism spectrum disorder (ASD, purple) and Schizophrenia (orange). (e) Violin plots showing a list of genes differentially expressed between Pvalb + neurons in TRN compared and Pvalb + neurons in the four other brain regions including hippocampus (HP), secondary motor cortex (M2), somatosensory cortex (SCX), and striatum (STR). (f) Violin plots confirming the TRN-enriched gene list as shown in panel (e) in additional brain regions using the mousebrain.org datasets. CB: cerebellum; Hypoth: hypothalamus; MBd: medial basal nucleus dorsal part; MBV: medial basal nucleus ventral part; SC: spinal cord; Thal: thalamus. (g) Violin plots showing selected differentially expressed disease-risk genes compared to the Pvalb + neurons in the other four brain regions as indicated. HP: hippocampus; M2: secondary motor cortex; SCX: somatosensory cortex; STR: striatum. For (e) and (g), n HP =90, n M2 =97, n SCX =116, n STR =13, n TRN =671 cells; For (f), n CA1 =136, n CB =477, n Hypoth =156, n MBb =331, n MBv =209, n Medulla =121, n Pons =199, n SC =69, n Thal =54, n TRN =501 cells. The violin plots width is based off of a Gaussian kernel density estimate of the data (estimated by the standard density function in R with default parameters), scaled to have maximum width equal to 1.

Article Snippet: RNAscope probes used were Mm-Spp1-C1 (ACDBio, USA, #435191, NM_001204201.1, region 2-1079), Mm-Ecel1-C3 (ACDBio, USA, #475331-C3, NM_001277925.1, region 879-1992), and Mm-Pvalb-C4 (ACDBio, USA, #421931-C4, NM_013645.3, region 2*885).

Techniques: CRISPR, In Vivo, Knock-Out, Expressing, Labeling

Journal: Nature

Article Title: Distinct subnetworks of the thalamic reticular nucleus

doi: 10.1038/s41586-020-2504-5

Figure Lengend Snippet: (a) Representative current-clamp recording traces of Spp1 + like (‘Spp1’, magenta) and Ecel1 + like (‘Ecel1’, green) neurons held at different membrane potentials. The trace with the maximum number of bursts was selected for measuring different burst properties and calculating the Z-score (shown in the right). (b) Plot showing confidence interval ellipses for classifying Spp1 + like (‘Spp1’) and Ecel1 + like (‘Ecel1’) neurons based on the AHP and the number of rebound bursts. (c) Representative rebound burst traces of recorded neurons after knocking out different sets of genes via CRISPR-Cas9 gene editing. Traces show rebound bursting activity changes in response to hyperpolarizing current step injections. TRN neurons exhibited distinct changes in their firing patterns after knockout of different gene groups. (d) Radar plots of 5 electrophysiological parameters illustrated in (a), showing the deviation of perturbed group to the control after knocking out sets of genes in the pooled approach. Positive changes show an increase towards a parameter, while negative changes show a decrease when compared to control. Green line indicates deviations in Ecel1 + like neurons and color shades indicates deviations in Spp1 + like neurons. (e) Summary of the maximum number of rebound bursts of TRN neurons elicited by comparable hyperpolarizing current step injection as described in after different sets of genes were knocked out0 in the pooled approach in Spp1 + like (‘Spp1’) vs. Ecel1 + like (‘Ecel1’) neurons (‘Spp1’ Pool1, p = 4.8742×10 7 ; Pool3, p=0.0033; Pool5, p=0.0088; Pool7, p=0.0065. ‘Ecel1’ Pool3, p=0.0081; Pool7, p=0.023, two-sided unpaired t-test). Bars represent the mean ± SEM. For (a)-(e): ‘Spp1’ Ighe n=12, Pool1 n=12, Pool2 n=9, Pool3 n=13, Pool4 n=9, Pool5 n=10, Pool6 n=9, and Pool7 n=10 cells; ‘Ecel1’ Ighe n=9, Pool1 n=12, Pool2 n=13, Pool3 n=10, Pool4 n=8, Pool5 n=10, Pool6 n=8, and Pool7 n=9 cells from 24 mice (3 mice per pool).

Article Snippet: RNAscope probes used were Mm-Spp1-C1 (ACDBio, USA, #435191, NM_001204201.1, region 2-1079), Mm-Ecel1-C3 (ACDBio, USA, #475331-C3, NM_001277925.1, region 879-1992), and Mm-Pvalb-C4 (ACDBio, USA, #421931-C4, NM_013645.3, region 2*885).

Techniques: Membrane, CRISPR, Activity Assay, Knock-Out, Injection

Journal: Nature

Article Title: Distinct subnetworks of the thalamic reticular nucleus

doi: 10.1038/s41586-020-2504-5

Figure Lengend Snippet: (a) Representative rebound burst traces of recorded neurons after knocking out different individual genes from Pool3 via CRISPR/Cas9 gene editing. Knocking out of Kcnd2 recapitulates the effects of Pool#3. (b) Radar plots for Pool3 individual gene. Top: Changes in Spp1 + like (‘Spp1’) neurons, pink line showing the effect of the Pool3 gene knock out and color shades showing the effect produced by individual gene knock out. Bottom: Changes in Ecel1 + like (‘Ecel1’) neurons, green line showing the Pool3 gene radar plot and color shades showing the changes produced by individual gene knockout. Kcnd2 knockout closely recapitulates the effect of Pool3 in both populations. (c) Summary of the maximum number of rebound bursts of TRN neurons elicited by comparable protocols after individual genes from Pool3 were knockout in Spp1 + like (‘Spp1’) vs. Ecel1 + like (‘Ecel1’) neurons (‘Spp1’ Kcnd2, p=0.0095. ‘Ecel1’ Kcng1, p=0.0088; Kcnd2, p=0.019, two-sided unpaired t-test). Bars represent the mean ± SEM. For (a)-(c), ‘Spp1’ Kcng1 n=6, Kcnc3 n=6, Kcng4 n=5, Kcnip1 n=7, Kcnd2 n=7; ‘Ecel1’ Kcng1 n=10, Kcnc3 n=8, Kcng4 n=9, Kcnip1 n=11, Kcnd2 n=11. (d) Schematics of the analysis for on-target and off-target efficiency. Upper: analysis flowchart. WGA: whole genome amplification; NGS: next generation sequencing. Lower: schematics of sgRNA design and primers for on-target analysis for Kcnd2 knockout. Five sgRNA were designed in Exon2, Exon3, and Exon4. As the length spanned by the leftmost sgRNA and the rightmost sgRNA exceeds the NGS analysis limit, nested PCR combined with Sanger sequencing was used for on-target efficiency analysis. Primers for the nested PCR are shown as black arrows in Exon1 and Exon5. (e) Bar chart showing the on-target efficiency (5 sgRNA pooled) analyzed by nested PCR and Sanger sequencing (control: n=96 nuclei, viral injected: n=384 nuclei) and off-target rate for the top predicted (see ) off-target loci of each sgRNA analyzed by NGS (n=1600 cells and 72000 nuclei). Predicted off-target sequences are shown with mismatched bases in lower case. Bar plots represent maximum likelihood estimation (MLE) and upper Wilson score intervals, no raw data point applicable 69 .

Article Snippet: RNAscope probes used were Mm-Spp1-C1 (ACDBio, USA, #435191, NM_001204201.1, region 2-1079), Mm-Ecel1-C3 (ACDBio, USA, #475331-C3, NM_001277925.1, region 879-1992), and Mm-Pvalb-C4 (ACDBio, USA, #421931-C4, NM_013645.3, region 2*885).

Techniques: CRISPR, Knock-Out, Produced, Gene Knockout, Whole Genome Amplification, Next-Generation Sequencing, Nested PCR, Sequencing, Injection

Journal: Nature

Article Title: Distinct subnetworks of the thalamic reticular nucleus

doi: 10.1038/s41586-020-2504-5

Figure Lengend Snippet: (a) TRN labeled retrogradely with injection in FO (upper) and HO (lower) thalamic nuclei. Left: coronal sections showing TRN ( Pvalb , PV) neurons retrogradely-labeled by Gamma6-mCherry (red). Right: Zoom-in views of merged and single-color images of boxed area in the left panel. Scale bar: 100μm. (b) Traces showing differential firing activity changes in TRN neurons in response to hyperpolarizing current-step injections after Gamma6 viral injections in FO vs. HO thalamic nuclei, which retrogradely labels preferentially Spp1 + and Ecel1 + neurons respectively. (c) Gamma6 significantly reduced the number of rebound bursts in TRN neurons (FO control 6.1 ± 1.6 bursts vs. Gamma6 1.7 ± 0.8 bursts, p=0.0001; HO control 1.1 ± 0.26 bursts vs. Gamma6 0.3 ± 0.1 bursts, p=0.0081, two-sided unpaired t-test). Mean ± SD and raw data points. For (a), (b), and (c), FO n control =14 and n Gamma6 =18 neurons collected from 4 animals, HO n control =12 and n Gamma6 =11 neurons collected from 4 animals. (d) Left: normalized power spectrum. Right: percentage of power in delta rhythm. Gamma6 expressed in TRN neurons labeled retrogradely from FO led to reduction in the power of delta rhythms (1-4 Hz) (FO: p=0.0045; HO: p=0.75, two-sided unpaired t-test, NS: not significant), (e) significant reduction in spindle density (FO: p=0.038; HO: p=0.96, two-sided Wilcoxon rank-sum test), and (f) decreased spindle length, but increased spindle length when expressed in TRN neurons labeled retrogradely from HO somatosensory thalamic nucleus (Kolmogorov-Smirnov test). Insert: Representative traces of sleep spindles. For data in (d), (e), and (f), FO n control =8, n Gamma6 =8, HO n control =7, n Gamma6 =8 animals. Box plots represent minima, 25th, 50th, 75th percentiles, maxima, and raw data points.

Article Snippet: RNAscope probes used were Mm-Spp1-C1 (ACDBio, USA, #435191, NM_001204201.1, region 2-1079), Mm-Ecel1-C3 (ACDBio, USA, #475331-C3, NM_001277925.1, region 879-1992), and Mm-Pvalb-C4 (ACDBio, USA, #421931-C4, NM_013645.3, region 2*885).

Techniques: Labeling, Injection, Activity Assay