70 µl ssc 0.1x (diluted with rnase and dnase free mq water from 20x ssc)  (Millipore)

Journal: Nature Communications

Article Title: Miniature spatial transcriptomics for studying parasite-endosymbiont relationships at the micro scale

doi: 10.1038/s41467-023-42237-y

Figure Lengend Snippet: A 3D model of the UMIs and unique genes per spot across worm sample BM2. Magnification 20x. A: Anterior, P: Posterior, arrow indicates first to last section through the worm. B UMAP showing the four clusters of Brugia malayi transcriptome data. C Spatial distribution of the four clusters on tissue sections from B. malayi samples. Magnification 20x. Sample names are to the left of each row of section images and section names are below each section image. D Percent of differentially expressed marker genes per cluster enriched for specific tissue regions. E Significantly overrepresented functional terms for each cluster were identified using a two-sided Fisher’s exact test (FDR < 0.05).

Article Snippet: Pepsin was removed and sections washed with 70 µL SSC 0.1X (diluted with RNase and DNase free MQ water from 20X SSC (Sigma-Aldrich, PN: S66391L)). cDNA reaction mix was prepared with 16 µL 5X FS buffer (Invitrogen, PN: Y02321), 4 µL 0.1 M DTT (Invitrogen, PN: Y00147), 33.3 µL RNase and DNase free MQ water, 8 µL diluted Actinomycin D (10 µL Actinomycin D (Sigma-Aldrich, PN: A1410) in 90 µL RNase and DNase free MQ water), 4 µL NEB BSA (20 mg/mL) (Molecular Biology Grade, New England BioLabs, Catalog # B9000)), dNTP mix (100 µL dATP Solution (100 mM) (Thermo Scientific, Cat No. R0141), 100 µL dGTP (100 mM) (Thermo Scientific, Cat No. R0161), 100 µL dTTP (100 mM) (Thermo Scientific, Cat No. R0171), 10 µL diluted dCTP (5 µL dCTP (100 mM) (Thermo Scientific, Cat No. R0151) in 20 µL RNase and DNase free MQ water), 690 µL RNase and DNase free MQ water), 2 µL diluted Cy3-dCTP (5 µL Cy3-dCTP (PerkinElmer, PN: NEL576001EA) in 20 µL RNase and DNase free MQ water), 4 µL RNaseOUT (Invitrogen, PN: 100000840), and 8 µL SuperScript III (Invitrogen, Cat No. 18080085) per well and pre-warmed at 42 ◦ C. Seventy µL of pre-warmed, 42 ◦ C cDNA reaction mix was added and incubated overnight at 42 ◦ C on a Thermoblock (ThermMixer with Thermoblock, Eppendorf) with a heated lid (ThermoTop, Eppendorf) for cDNA synthesis.

Techniques: Marker, Functional Assay

Journal: Nature Communications

Article Title: Miniature spatial transcriptomics for studying parasite-endosymbiont relationships at the micro scale

doi: 10.1038/s41467-023-42237-y

Figure Lengend Snippet: A Dotplot depicting the expression of B. malayi cluster marker genes across the different clusters. Average expression values indicate the average UMI counts (normalized) per spot across all spots in each cluster. Percent expressed values indicate the percentage of spots within each cluster that express each gene. B – F Spatial expression of cluster marker genes in 3D through worm sample BM2 and expression distribution across each cluster in violin plot. Magnification 20x. A: Anterior, P: Posterior, arrow indicates first to last section through the worm.

Article Snippet: Pepsin was removed and sections washed with 70 µL SSC 0.1X (diluted with RNase and DNase free MQ water from 20X SSC (Sigma-Aldrich, PN: S66391L)). cDNA reaction mix was prepared with 16 µL 5X FS buffer (Invitrogen, PN: Y02321), 4 µL 0.1 M DTT (Invitrogen, PN: Y00147), 33.3 µL RNase and DNase free MQ water, 8 µL diluted Actinomycin D (10 µL Actinomycin D (Sigma-Aldrich, PN: A1410) in 90 µL RNase and DNase free MQ water), 4 µL NEB BSA (20 mg/mL) (Molecular Biology Grade, New England BioLabs, Catalog # B9000)), dNTP mix (100 µL dATP Solution (100 mM) (Thermo Scientific, Cat No. R0141), 100 µL dGTP (100 mM) (Thermo Scientific, Cat No. R0161), 100 µL dTTP (100 mM) (Thermo Scientific, Cat No. R0171), 10 µL diluted dCTP (5 µL dCTP (100 mM) (Thermo Scientific, Cat No. R0151) in 20 µL RNase and DNase free MQ water), 690 µL RNase and DNase free MQ water), 2 µL diluted Cy3-dCTP (5 µL Cy3-dCTP (PerkinElmer, PN: NEL576001EA) in 20 µL RNase and DNase free MQ water), 4 µL RNaseOUT (Invitrogen, PN: 100000840), and 8 µL SuperScript III (Invitrogen, Cat No. 18080085) per well and pre-warmed at 42 ◦ C. Seventy µL of pre-warmed, 42 ◦ C cDNA reaction mix was added and incubated overnight at 42 ◦ C on a Thermoblock (ThermMixer with Thermoblock, Eppendorf) with a heated lid (ThermoTop, Eppendorf) for cDNA synthesis.

Techniques: Expressing, Marker

Journal: Nature Communications

Article Title: Miniature spatial transcriptomics for studying parasite-endosymbiont relationships at the micro scale

doi: 10.1038/s41467-023-42237-y

Figure Lengend Snippet: A Dotplot depicting expression of glycolytic genes across the different clusters. Average expression values indicate the average UMI counts (normalized) per spot across all spots in each cluster. Percent expressed values indicate the percentage of spots within each cluster that express each gene. B – D 3D model of gene expression in sample BM2, 2D gene expression across select sections from all 3 samples (normalized UMI counts per spot), and the gene expression distribution (normalized UMI counts per spot) across the clusters in a violin plot for glycolytic genes Bma-aldo-1 ( B ), Bm5699 ( C ), and Bma-ldh-1 ( D ). Magnification 20x. A: Anterior, P: Posterior, arrow in each 3D plot indicates first to last section through the worm. Sample names are to the left of each row of section images and section numbers are below each section image.

Article Snippet: Pepsin was removed and sections washed with 70 µL SSC 0.1X (diluted with RNase and DNase free MQ water from 20X SSC (Sigma-Aldrich, PN: S66391L)). cDNA reaction mix was prepared with 16 µL 5X FS buffer (Invitrogen, PN: Y02321), 4 µL 0.1 M DTT (Invitrogen, PN: Y00147), 33.3 µL RNase and DNase free MQ water, 8 µL diluted Actinomycin D (10 µL Actinomycin D (Sigma-Aldrich, PN: A1410) in 90 µL RNase and DNase free MQ water), 4 µL NEB BSA (20 mg/mL) (Molecular Biology Grade, New England BioLabs, Catalog # B9000)), dNTP mix (100 µL dATP Solution (100 mM) (Thermo Scientific, Cat No. R0141), 100 µL dGTP (100 mM) (Thermo Scientific, Cat No. R0161), 100 µL dTTP (100 mM) (Thermo Scientific, Cat No. R0171), 10 µL diluted dCTP (5 µL dCTP (100 mM) (Thermo Scientific, Cat No. R0151) in 20 µL RNase and DNase free MQ water), 690 µL RNase and DNase free MQ water), 2 µL diluted Cy3-dCTP (5 µL Cy3-dCTP (PerkinElmer, PN: NEL576001EA) in 20 µL RNase and DNase free MQ water), 4 µL RNaseOUT (Invitrogen, PN: 100000840), and 8 µL SuperScript III (Invitrogen, Cat No. 18080085) per well and pre-warmed at 42 ◦ C. Seventy µL of pre-warmed, 42 ◦ C cDNA reaction mix was added and incubated overnight at 42 ◦ C on a Thermoblock (ThermMixer with Thermoblock, Eppendorf) with a heated lid (ThermoTop, Eppendorf) for cDNA synthesis.

Techniques: Expressing

Journal: Nature Communications

Article Title: Miniature spatial transcriptomics for studying parasite-endosymbiont relationships at the micro scale

doi: 10.1038/s41467-023-42237-y

Figure Lengend Snippet: A UMAP showing spots with presence ( Wolbachia + /W + ) or absence ( Wolbachia -/W-) of Wolbachia . Dotted lines outline the different clusters from Fig. . B Volcano plot showing the differentially expressed genes in Wolbachia + versus Wolbachia - spots (green: average log fold change <0.4 and adjusted p value < 0.05, orange: average log fold change > 0.4 and adjusted p -value < 0.05). Differential ex p ression analysis used the Wilcoxon Rank Sum test and adjusted p values ( p) were estimated with a two-sided alternative hypothesis. C – D 3D model and 2D images of gene expression in sample BM2 (sections BM2_4 and BM2_9) for two differentially expressed genes, Bma-snx-1 ( C ) and Bma-ned-8 ( D ), between Wolbachia + (W + ) and Wolbachia - (W-) spots. Magnification 20x. A: Anterior, P: Posterior, arrow indicates first to last section through the worm.

Article Snippet: Pepsin was removed and sections washed with 70 µL SSC 0.1X (diluted with RNase and DNase free MQ water from 20X SSC (Sigma-Aldrich, PN: S66391L)). cDNA reaction mix was prepared with 16 µL 5X FS buffer (Invitrogen, PN: Y02321), 4 µL 0.1 M DTT (Invitrogen, PN: Y00147), 33.3 µL RNase and DNase free MQ water, 8 µL diluted Actinomycin D (10 µL Actinomycin D (Sigma-Aldrich, PN: A1410) in 90 µL RNase and DNase free MQ water), 4 µL NEB BSA (20 mg/mL) (Molecular Biology Grade, New England BioLabs, Catalog # B9000)), dNTP mix (100 µL dATP Solution (100 mM) (Thermo Scientific, Cat No. R0141), 100 µL dGTP (100 mM) (Thermo Scientific, Cat No. R0161), 100 µL dTTP (100 mM) (Thermo Scientific, Cat No. R0171), 10 µL diluted dCTP (5 µL dCTP (100 mM) (Thermo Scientific, Cat No. R0151) in 20 µL RNase and DNase free MQ water), 690 µL RNase and DNase free MQ water), 2 µL diluted Cy3-dCTP (5 µL Cy3-dCTP (PerkinElmer, PN: NEL576001EA) in 20 µL RNase and DNase free MQ water), 4 µL RNaseOUT (Invitrogen, PN: 100000840), and 8 µL SuperScript III (Invitrogen, Cat No. 18080085) per well and pre-warmed at 42 ◦ C. Seventy µL of pre-warmed, 42 ◦ C cDNA reaction mix was added and incubated overnight at 42 ◦ C on a Thermoblock (ThermMixer with Thermoblock, Eppendorf) with a heated lid (ThermoTop, Eppendorf) for cDNA synthesis.

Techniques: Expressing

Journal: Nature Communications

Article Title: Miniature spatial transcriptomics for studying parasite-endosymbiont relationships at the micro scale

doi: 10.1038/s41467-023-42237-y

Figure Lengend Snippet: A Violin plots of the B. malayi UMIs and unique genes per capture spot across different treated worm tissue sample sections. B Scatter plot of the average B. malayi gene expression between consecutive treated worm sections (T1_4 and T1_5) across all genes ( r = 0.92, p < 0.05). The statistical test is based on the Pearson’s product moment correlation coefficient (r) with 95% confidence intervals and p -values ( p) were estimated with two-sided alternative hypothesis. C Violin plots of the Wolbachia unique molecules (UMIs) per spot and unique genes per spot in control and treated worm sample sections. D Violin plots of the B. malayi unique molecules (UMIs) per spot and unique genes per spot in control and treated worm sample sections in Wolbachia + (W + ) and Wolbachia- (W-) spots. E Dotplot depicting the expression of select B. malayi genes in Wolbachia+ spots in treated worms (W + _T), Wolbachia+ spots in control worms (W + _C), Wolbachia- spots in treated worms (W-_T), and Wolbachia- spots in control worms (W-_C). Average expression values indicate the average UMI counts (normalized) per spot across all spots in each cluster. Percent expressed values indicate the percentage of spots within each cluster that express each gene. F 2D images of Bma-snx-1 and Bma-ned-8 gene expression in sample section T2_1 between Wolbachia + (W + ) and Wolbachia - (W-) spots. Magnification 20x.

Article Snippet: Pepsin was removed and sections washed with 70 µL SSC 0.1X (diluted with RNase and DNase free MQ water from 20X SSC (Sigma-Aldrich, PN: S66391L)). cDNA reaction mix was prepared with 16 µL 5X FS buffer (Invitrogen, PN: Y02321), 4 µL 0.1 M DTT (Invitrogen, PN: Y00147), 33.3 µL RNase and DNase free MQ water, 8 µL diluted Actinomycin D (10 µL Actinomycin D (Sigma-Aldrich, PN: A1410) in 90 µL RNase and DNase free MQ water), 4 µL NEB BSA (20 mg/mL) (Molecular Biology Grade, New England BioLabs, Catalog # B9000)), dNTP mix (100 µL dATP Solution (100 mM) (Thermo Scientific, Cat No. R0141), 100 µL dGTP (100 mM) (Thermo Scientific, Cat No. R0161), 100 µL dTTP (100 mM) (Thermo Scientific, Cat No. R0171), 10 µL diluted dCTP (5 µL dCTP (100 mM) (Thermo Scientific, Cat No. R0151) in 20 µL RNase and DNase free MQ water), 690 µL RNase and DNase free MQ water), 2 µL diluted Cy3-dCTP (5 µL Cy3-dCTP (PerkinElmer, PN: NEL576001EA) in 20 µL RNase and DNase free MQ water), 4 µL RNaseOUT (Invitrogen, PN: 100000840), and 8 µL SuperScript III (Invitrogen, Cat No. 18080085) per well and pre-warmed at 42 ◦ C. Seventy µL of pre-warmed, 42 ◦ C cDNA reaction mix was added and incubated overnight at 42 ◦ C on a Thermoblock (ThermMixer with Thermoblock, Eppendorf) with a heated lid (ThermoTop, Eppendorf) for cDNA synthesis.

Techniques: Expressing