Journal: bioRxiv

Article Title: Loss of Ezh2 in the medial ganglionic eminence alters interneuron fate, cell morphology and gene expression profiles

doi: 10.1101/2023.09.06.556544

Figure Lengend Snippet: A. Visualization of quality control (QC) metrics before (Pre-QC) and after (Post-QC) filtering of outliers. The number of RNA reads (nCount_RNA), mitochondrial percentage (percent.mt), the number of ATAC fragments (nCount_ATAC), nucleosome signal (nucleosome_signal), and the TSS enrichment score (TSS.enrichment) are shown. B. UMAP plots of Multiome data separated by age (E12.5, E15.5 and combined) and modality (RNA, ATAC and integrated RNA+ATAC), with putative cell clusters. WT = blue, Het = red, KO = green.

Article Snippet: We generated single nuclei suspensions from E12.5 and E15.5 MGE and used the 10x Genomics Multiome kit to define the gene expression profile and chromatin accessibility within individual cells.

Techniques:

Journal: bioRxiv

Article Title: Loss of Ezh2 in the medial ganglionic eminence alters interneuron fate, cell morphology and gene expression profiles

doi: 10.1101/2023.09.06.556544

Figure Lengend Snippet: A. Uniform Manifold Approximation and Projection (UMAP) plots of E12.5 and E15.5 integrated single cell RNA- and ATAC-seq (Multiome) dataset via weighted nearest neighbor (WNN), annotated by age and genotype (left and middle) or putative cell clusters (right). Labels for putative cell clusters are listed above the UMAP. B. Markers for radial glia cells ( Nes ), cycling GE progenitors ( Ascl1 ) and postmitotic immature neurons ( Dcx , Rbfox3 ), with general trajectory confirmed by pseudotime. C. SST is enriched in E15.5 KO MGE postmitotic neurons compared WT MGE. D. Enrichment of Mef2c and Maf , two genes predictive of PV-fated interneurons, in E15.5 WT MGE compared to KO. E. Enrichment of Mef2c and Maf binding motifs in E15.5 WT MGE compared to KO. F. E15.5 integrated RNA and ATAC dataset annotated by genotype (left) and age (right). G. Differential abundance (DA) analysis using DA-seq reveals that SST + cells are located in KO-enriched clusters (red) whereas Mef2c - and Maf -expressing cells are located in WT-enriched clusters (blue). DA score threshold of +/- 0.7 used for significant enrichment of DA cells in DA-seq analysis. H. Clusters 3, 4, 5 and 7 from panel A that are enriched for PV- and SST-fated cells. I. Volcano plots depicting genes enriched in WT or KO MGE at E12.5 (top) and E15.5 (bottom). Thresholds used were Log 2 fold change (FC) > ± 0.2 and a false discovery rate (FDR) of Log 10 P < 10 - . J. E15.5 integrated RNA and ATAC dataset annotated by genotype (top left) and putative cell clusters (bottom left). The top gene enriched in the cluster harboring SST + cells (Cluster 10, arrow) is Pde1a . Pde1a is expressed in many SST+ interneuron subtypes in the adult mouse (each row is a mature interneuron subtype), but excluded from PV+ interneurons (right, adapted from the Allen Brain Map Transcriptomics Explorer).

Article Snippet: We generated single nuclei suspensions from E12.5 and E15.5 MGE and used the 10x Genomics Multiome kit to define the gene expression profile and chromatin accessibility within individual cells.

Techniques: Binding Assay, Expressing

Journal: bioRxiv

Article Title: Single-Nucleus Neuronal Transcriptional Profiling of Male C. elegans Uncovers Regulators of Sex-Specific and Sex-Shared Behaviors

doi: 10.1101/2024.12.12.628226

Figure Lengend Snippet: (A) Representative image of pan-neuronal GFP tagged him-8 males ( Prgef-1::his-58::GFP;him-8) . (B) 6-well tissue culture inserts with modified filters were used to separate sexes in C. elegans, achieving an average ∼95% purity for both male and hermaphrodite fractions. (C) Overview of nuclei isolation and sequencing. Pan-neuronal histone GFP-tagged him-8 males and hermaphrodites were separated, nuclei were isolated, FACS sorted for Hoescht and DAPI positive nuclei, followed by barcoding, cDNA amplification, and library preparation using 10X genomics chromium X, then Illumina RNA sequenced. (D) UMAP of 61,936 male-specific neurons that passed quality control. (E) Feature plot of pkd-2, clec-164, flp-17, and Y70G10A.2 expression (known markers expressed in CEM, HOB, and ray neurons).

Article Snippet: Single nuclei suspension samples were then provided to the Princeton Genomics Core for 10X Genomics Chromium X barcoding, cDNA amplification, library preparation and Illumina next generation sequencing, as described in St. Ange and Weng et al. 2024 .

Techniques: Modification, Isolation, Sequencing, Amplification, Control, Expressing

Journal: bioRxiv

Article Title: Resource: Scalable whole genome sequencing of 40,000 single cells identifies stochastic aneuploidies, genome replication states and clonal repertoires

doi: 10.1101/411058

Figure Lengend Snippet: a Cross contamination test for new primer wash routine. A less aggressive wash routine using Tween-20 was tested to replace the primer spotting wash routine using Sciclean as aggressive repetitive Sciclean washes were stripping the nozzle coating and causing issues with drop stability. The new Tween-20 routine was compared to the Sciclean washes by alternating spotting of human and salmon sperm gDNA with a wash step in between and then checking for cross contaminating reads to demonstrate the new wash routine acceptably prevented carryover between wells. The control is human cells dispensed into wells to show typical levels of expected human and salmon reads when primers were dispensed using the Sciclean wash routine. Box plots of fraction of total reads aligned to human and salmon show median and quartiles, the whiskers show the remaining distribution, and dots indicate outliers. b Relating to , the effect of lysis time on coverage breadth of merged single-cell genomes. Bootstrap sampling of single-cell GM18507 libraries prepared using a 2 hour and overnight cold lysis conditions; DLP+ 2 hours (n = 148), DLP+ overnight (n = 133), MF-DLP (n = 122). Single-cell libraries were downsampled to a similar median coverage depth. Box plots show median and quartiles, the whiskers show the remaining distribution, and dots indicate outliers. Lorenz curves shows coverage uniformity for merged single-cell genomes. Curves are median merged genomes. Experimental condition and number of merged cells are indicated in the plot. Dotted black line indicates perfectly uniform genome. c Effect of Tn5 concentration and PCR cycles time on coverage breadth of merged single-cell genomes. Bootstrap sampling of single-cell GM18507 libraries prepared using a range of Tn5 concentrations and PCR indexing cycles on the open-array and compared to the MF-DLP dataset (7); DLP+ 2.2 nl Tn5, 8 PCR (n =188), 3.5 nl Tn5, 8 PCR (n = 190), 6.5 nl Tn5, 8 PCR (n = 197), 2.2 nl Tn5, 11 PCR (n = 198), and MF-DLP (7) (n = 122). Single-cell libraries were downsampled to a similar median mean coverage depth. Coverage depth and coverage breadth are shown in boxplots. d Effect of cell dispensing method on total mapped reads, with active selection (cellenONE, spotted in a block of wells or a scatter pattern) or passive limiting dilution dispensing. Black lines show median. e Effect of on-chip storage of isolated cells and nuclei. Quality score of cells and nuclei that were dispensed into nanowells and stored either overnight or for 63 days prior to lysis and library construction. Dots represent individual cells/nuclei. Black lines show median. f Effect of protease concentration on cells. Quality scores of single-cell libraries built with a low, medium, or high concentration of protease in the lysis buffer and lysed for either 2 or 19 hours, followed by library construction with a range of protease concentrations. g Relating to , h comparisons between MF-DLP and DLP+ with two Tn5 concentrations, boxplots show additional sequencing metrics (total reads, fraction unmapped reads, coverage depth). Box plots show median and quartiles, the whiskers show the remaining distribution, and dots indicate outliers. The top column labels state the numbers of cells per condition. Colors represent experimental condition.

Article Snippet: For a subset of samples (GM18507, SA501X11XB00529, SA611X3XB00821), nuclei were prepared from single cell suspensions by doubling the volume of the cells with Nuclei EZ lysis buffer (Sigma) before staining, to compare nuclei data to cell data.

Techniques: Stripping Membranes, Lysis, Sampling, Concentration Assay, Selection, Blocking Assay, Isolation, Sequencing