experimental workflows  (Novartis)

Journal: The Journal of comparative neurology

Article Title: Vascular Development of Fetal and Postnatal Neocortex of the Pig, the European Wild Boar Sus scrofa.

doi: 10.1002/cne.70011

Figure Lengend Snippet: FIGURE 4 Quantitative assessment of vasculature. (A) Experimental workflow from dissection to filament tracing (Imaris). The raw confocal image was processed with a threshold filter and a masking algorithm. Next, the “surface” tool was used to create a surface for volume measurement and masking algorithm. The “filament” tool yielded a semiautomatic layout of the vessel pattern, which has in all cases been manually refined. Vascular parameters were addressed in four laminar compartments. If present, the SP was included in the IZ/WM compartment. (B) Volume of all vessels compared with the total sample volume. (C) Vessel branching points were calculated per 100 µm. (D) The length of vessels was set in relation to the total sample volume. (E) The average vessel segment area. Vessel sections between two branching points were defined as segments. The numbers in the bars represent the number of nonadjacent regions of interest that were assessed for this compartment. At P30, no sufficient SVZ/VZ area could be found suitable for reliable reconstruction of that compartment. n. a., not applicable. Error bars represent the mean ± SEM. Scale bars: 40 µm in A.

Article Snippet: See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense FIGURE 4 Quantitative assessment of vasculature. (A) Experimental workflow from dissection to filament tracing (Imaris).

Techniques: Dissection

Journal: Genome Biology

Article Title: scifi-ATAC-seq: massive-scale single-cell chromatin accessibility sequencing using combinatorial fluidic indexing

doi: 10.1186/s13059-024-03235-5

Figure Lengend Snippet: Scifi-ATAC-seq combines pre-indexing with droplet-based scATAC-seq. a Schematic of regular droplet-based 10X Genomics scATAC-seq experimental workflow. b Schematic of scifi-ATAC-seq experimental workflow. c Distributions of the proportion of Tn5 integration sites within the promoter regions, encompassing the 2-kb flanking regions around gene transcription start sites (TSSs). d Distributions of the proportion of Tn5 integration sites within peaks per nucleus. e Distribution of unique Tn5 integration sites per nucleus. f Number of nuclei that passed quality control thresholds. g – i Scatterplot displaying the number of reads per cell classified as B73 or Mo17, color-coded by genotype classification. g 16 k input B73/Mo17 scATAC-seq; h 100 k scifi-ATAC-seq; i 200 k scifi-ATAC-seq. Median contamination rate: the median cross-contamination rate, attributed to index hopping, among all predicted singlets. j UMAP of all nuclei ( n = 98,424). Nuclei are colored by their predicted cell type. k Pseudobulk cell type Tn5 integration site coverage around the phloem precursor marker ZmSMXL3 . Vas. par. precursor: Vascular parenchyma precursor. l Pseudobulk cell type Tn5 integration site coverage for and UMAP embeddings overlaid with gene chromatin accessibility around the ZmSMXL3 gene across all datasets. N , number of phloem nuclei

Article Snippet: Fig. 1 Scifi-ATAC-seq combines pre-indexing with droplet-based scATAC-seq. a Schematic of regular droplet-based 10X Genomics scATAC-seq experimental workflow. b Schematic of scifi-ATAC-seq experimental workflow. c Distributions of the proportion of Tn5 integration sites within the promoter regions, encompassing the 2-kb flanking regions around gene transcription start sites (TSSs). d Distributions of the proportion of Tn5 integration sites within peaks per nucleus. e Distribution of unique Tn5 integration sites per nucleus. f Number of nuclei that passed quality control thresholds. g – i Scatterplot displaying the number of reads per cell classified as B73 or Mo17, color-coded by genotype classification. g 16 k input B73/Mo17 scATAC-seq; h 100 k scifi-ATAC-seq; i 200 k scifi-ATAC-seq.

Techniques: Marker

Journal: RSC Medicinal Chemistry

Article Title: Enabling synthesis in fragment-based drug discovery (FBDD): microscale high-throughput optimisation of the medicinal chemist's toolbox reactions †

doi: 10.1039/d3md00495c

Figure Lengend Snippet: Fig. 3

Article Snippet: In order to mitigate the guesswork needed and reduce the amount of time and material required to optimise these cross-couplings, we have established an in-house high-throughput experimentation (HTE) workflow that all Astex chemists can use with minimal training ( Fig. 3 ).

Techniques: Software

Journal: EMBO Reports

Article Title: Resting natural killer cell homeostasis relies on tryptophan/ NAD + metabolism and HIF ‐1α

doi: 10.15252/embr.202256156

Figure Lengend Snippet: NK cell count, defined as CD45 + CD3 − NKp46 + CD49b + , in spleens of WT and HIF‐1α KO mice ( n = 2). Experimental design for in situ experiments. Heatmap of the downregulated metabolites from the untargeted metabolomics performed on six samples of freshly isolated NK cells from WT and HIF‐1α KO mice. Gene expression analysis of genes from the NAD—tryptophan pathway and key amino acid transporters from bulk RNA‐sequencing performed on six samples of freshly isolated NK cells from WT and HIF‐1α KO mice. The NAD—tryptophan pathway (Trp, tryptophan; Kyn, kynurenine; QA, quinolinic acid; NAAD, nicotinic acid adenine dinucleotide; NA, nicotinic acid; NAMN, nicotinic acid mononucleotide; NMN, nicotinamide mononucleotide; NAM, nicotinamide; MNA, methylnicotinamide; NAR, nicotinic acid riboside; NAD, nicotinamide adenine dinucleotide) and its key enzymes (IDO, indoleamine‐2,3‐dioxygenase; KMO, kynurenine 3‐monooxygenase; KYNU, kynureninase; HAAO, 3‐hydroxyanthranilate 3,4‐dioxygenase; QPRT, quinolinate phosphoribosyl‐transferase; NMNAT, NMN adenyl‐transferase; NAPRT, NA phosphoribosyl‐transferase; NADS, NAD synthase; NAMPT, NAM phosphoribosyl‐transferase; NRK, NR kinase) and analysis of metabolites quantification from untargeted (tryptophan, methyl‐quinoline and NAM) and targeted (NAAD, NA, NMN, MNA and kynurenine) metabolomics. NAD/NADH ratio ( n = 1). Data information: Statistical significance was determined by an unpaired Student's t ‐test. Bars represent mean values, error bars indicate the s.e.m., ( n ) represents the number of independent experiments, and each data point represents a biological sample from a mouse (A) or from NK cells pooled from 3 to 5 mice (D–F). Statistical significance is indicated as * P < 0.05, ** P < 0.01, and *** P < 0.001. Source data are available online for this figure.

Article Snippet: Samples were kept at −80°C until they were sent to Creative Proteomics who performed the sample preparation and the metabolomics experimental workflow.

Techniques: Cell Counting, In Situ, Isolation, Gene Expression, RNA Sequencing

Journal: EMBO Reports

Article Title: Resting natural killer cell homeostasis relies on tryptophan/ NAD + metabolism and HIF ‐1α

doi: 10.15252/embr.202256156

Figure Lengend Snippet: A, B NK cell, defined as CD45 + CD3 − NKp46 + CD49b + , (A) count and (B) frequency in bone marrow of WT and HIF‐1α KO mice ( n = 3). C, D NK cell (A) count and (D) frequency in liver of WT and HIF‐1α KO mice ( n = 3). E Gene expression analysis of genes from the NAD—tryptophan pathway and key amino acid transporters from bulk RNA‐sequencing performed on six samples of freshly isolated NK cells from WT and HIF‐1α KO mice. F The tryptophan pathway (Trp, tryptophan; NFK, N‐formylkynurenine; Kyn, kynurenine; KA, kynurenic acid; 3‐HK, 3‐hydroxykynurenine; 3‐HAA, 3‐hydroxyanthranilic acid; ACMS, 2‐amino‐3‐carboximuconate semialdehyde; AMS, aminomuconate semialdehyde; QA, quinolinic acid) and GSEA analysis of the RNA expression of its key enzymes (Ido, indoleamine‐2,3‐dioxygenase; Tdo, tryptophan‐2,3‐dioxygenase; Afmid, kynurenine formamidase; Kyat, kynurenine aminotransferase; Kmo, kynurenine 3‐monooxygenase; Kynu, kynureninase; Haao, 3‐hydroxyanthranilate 3,4‐dioxygenase; Acmsd, ACMS decarboxylase). G Analysis of quinolinic acid quantification from targeted metabolomics. H Analysis of mitochondrial ROS amount of freshly isolated NK cells from bone marrow (BM) and liver of WT and HIF‐1α KO mice ( n = 3). I Analysis of DNA damage in bone marrow (BM) and liver NK cells from WT and HIF‐1α KO mice by FACS measurement of γ‐H2AX ( n = 3). Data information: Statistical significance was determined by an unpaired Student's t ‐test. Bars represent mean values, error bars indicate the s.e.m., ( n ) represents the number of independent experiments, and each data point represents a biological sample from a mouse. Source data are available online for this figure.

Article Snippet: Samples were kept at −80°C until they were sent to Creative Proteomics who performed the sample preparation and the metabolomics experimental workflow.

Techniques: Gene Expression, RNA Sequencing, Isolation, RNA Expression