Journal: Nature

Article Title: CRISPR Screen in Regulatory T Cells Reveals Modulators of Foxp3

doi: 10.1038/s41586-020-2246-4

Figure Lengend Snippet: a) Representative flow cytometry analysis of the YFP + Treg population (gated on CD4 + cells) from the spleen and lymph nodes of Usp22 +/+ Foxp3 YFP-Cre WT or Usp22 fl/fl Foxp3 YFP-Cre KO mice. b) Statistical analysis of YFP MFI in CD4 + YFP + Tregs from the thymus (Thy), peripheral lymph nodes (pLN), and spleen (Spl) of Usp22 +/+ Foxp3 YFP-Cre WT or Usp22 fl/fl Foxp3 YFP-Cre KO mice. c) Statistical analysis of CD4 + YFP + Treg frequencies in Usp22 +/+ Foxp3 YFP-Cre WT or Usp22 fl/fl Foxp3 YFP-Cre KO mice, corresponding to panel b. d) Volcano plot for RNA sequencing of Usp22 RNP KO Tregs vs Rnf20 RNP KO murine Tregs. X-axis shows log2FoldChange (LFC). Y-axis shows the –log10 of the p-value as calculated by DESeq2. Genes downregulated in the Usp22 RNP KO compared to Rnf20 RNP KO are shown in red and genes upregulated are shown in blue defined by p-value <5e-3 and LFC > 0.8. Foxp3 (shown in green) trended down but did not reach significance. e) qPCR analysis of FOXP3 mRNA levels in human Tregs from 2 donors 8 days post-electroporation with Cas9 RNPs targeting NTC , FOXP3, USP22, RNF20 or both USP22 and RNF20. Normalized to the expression of β -ACTIN transcripts. Data are presented as mean ±SEM and are representative of at least two independent experiments. f) qPCR analysis of Foxp3 mRNA levels in mouse Tregs 4 and 8 days post-electroporation with Cas9 RNPs targeting NTC, Foxp3, Usp22, Rnf20 or both Usp22 and Rnf20. Normalized to the expression of β -actin transcripts. g) Western blot analysis of ubiquitinated histone 2A (H2AK119Ub; H2A-ub) and ubiquitinated histone 2B (H2BK120Ub; H2B-ub) from iTregs from Usp22 +/+ Foxp3 YFP-Cre WT or Usp22 fl/fl Foxp3 YFP-Cre KO mice. Gapdh was used as a loading control. Source data can be found in . h) Schematic of Foxp3 locus depicting PCR products used for ChIP-qPCR data shown in panel i and panel j. i) ChIP-qPCR data analysis for H2AK119Ub (H2A-ub) where primers amplified across the transcriptional start site (TSS) and the CNS1 enhancer region of the Foxp3 locus. Data are normalized to the input and are presented as mean ±SD. j) ChIP-qPCR data analysis for H2BK120Ub (H2B-ub) for PCR across the transcriptional start site (TSS) and across the CNS1 enhancer region of the Foxp3 locus. Data are normalized to the input and are presented as mean ±SD. k) Heatmap of ChIP-seq read density for Foxp3, Usp22, and Rnf20 at sites bound by Foxp3 (using previously published Foxp3 ChIP data ), ranked by highest to lowest Foxp3 binding signal. The corresponding log2 fold change (log2fc) for either H2BK120Ub or H2AK119Ub upon Usp22 or Rnf20 deletion at these sites are plotted on the right, with each biological replicate shown as an individual column. l) Average ChIP-seq read density of H2BK120Ub at Treg super enhancers in control versus Usp22-deficient Tregs. m) Co-occurrence analysis showing the natural log of the ratio of the observed number of overlapping regions over the expected values for sites that either gain or lose H2BK120Ub in Usp22-deficient Tregs against publicly available histone modifications H3K4me, H3K4me3 and H3K27ac as well as enhancer classes, as described in the Methods. n) Analysis of reciprocal regulation of Foxp3 by deubiquitinase Usp22 and E3 ubiquitin ligase Rnf20. YFP MFI of Tregs sorted from Usp22 +/+ Foxp3 YFP-Cre WT or Usp22 fl/fl Foxp3 YFP-Cre KO mice and then electroporated with either NT control (NTC-RNP) or Rnf20 RNP, corresponding with where Foxp3 MFI from the same experiment is shown. All data are presented as mean ±SEM, unless otherwise stated. ns indicates no significant difference, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. The exact sample sizes (n), p-values, statistical tests and number of times the experiment was replicated can be found in the “Statistics and Reproducibility” section.

Article Snippet: Tregs were collected from their culture vessels and centrifuged for 5 min at 300g, aspirated, and resuspended in the Lonza electroporation buffer P3 using 20 μl buffer per 200,000 cells.

Techniques: Flow Cytometry, RNA Sequencing Assay, Electroporation, Expressing, Western Blot, Amplification, ChIP-sequencing, Binding Assay

Journal: eLife

Article Title: A functional genetic toolbox for human tissue-derived organoids

doi: 10.7554/eLife.67886

Figure Lengend Snippet: ( a ) Efficiency quantification of different DNA delivery methods. EGFP-positive cells were quantified by flow cytometry 72 hr after transfection or transduction. N = 4 (or 3 for nucleofection EA104) different organoid lines were used for each condition. Error bars are plotted to show mean ± SEM. ( b ) Representative images showing DNA delivery efficiencies of different methods 72 hr after transfection/transduction. Top panel: widefield images; bottom panel: GFP channel. Scale bar denotes 100 μm. ( c ) T7 endonuclease assay showing the DNA cleavage efficiency of different CRISPR methods on the ACTB locus. Arrows denote lower bands generated by T7 endonuclease cutting. Left panel: schematic showing the different methods tested for introducing the Cas9 complex. cr/tr RNP, synthetic crispr/tracer RNA heterodimer with Cas9 RNP; ssRNP, single-strand synthetic guide RNA with Cas9 RNP. ( d ) Quantification of indels produced by the different CRISPR methods tested using ICE online analysis software ( https://ice.synthego.com/ ). N = 3 different organoid lines were used for each condition. Error bars are plotted to show mean ± SEM.

Article Snippet: 6 × 10 5 cells were suspended using Lonza Nucleofection P3 buffer, mixed with 6 μg of SOX9 reporter repair template plasmid.

Techniques: Flow Cytometry, Transfection, Transduction, CRISPR, Generated, Produced, Software

Journal: eLife

Article Title: A functional genetic toolbox for human tissue-derived organoids

doi: 10.7554/eLife.67886

Figure Lengend Snippet: ( a ) Schematic of the Organoid Easytag workflow. ssRNP and a circular plasmid repair template are nucleofected into dissociated cells at day 0. By day 3, cells have proliferated to become tiny colonies and are removed from the Matrigel and dissociated for selection by flow cytometry. EGFP + cells are re-plated sparsely (~1000–1500 cells/well of a 24-well plate) and grown until day 15 when organoids reach a sufficient size to be manually picked under a fluorescent microscope. Typically, 10–40 organoid colonies formed per ~1000 cells seeded. Organoids are picked into individual wells and passaged until sufficient cells are obtained for both genotyping and freezing down the line. Cells with red nuclei represent incorrectly targeted cells. Cells with white nuclei denote correctly targeted cells. ( b ) Schematic of repair template design for N terminal fusion mEGFP-ACTB gene targeting and the final product. Arrow shows the position of gRNA. E1, exon 1; E2, exon 2; 5′HA, 5′ homology arm; 3′HA, 3′ homology arm. ( c ) Representative flow cytometry results showing the percentage of EGFP cells 72 hr after nucleofection is performed. ( d ) Representative image showing mEGFP-ACTB organoid. DIC channel on the left and EGFP channel on the right. ( e ) Immunofluorescence of mEGFP-ACTB organoids. Blue: DAPI (nuclei); green: EGFP (ACTB fusion protein); red: SOX9 (lung progenitor marker). ( f ) Schematic of the AAVS1 targeting repair template design and the final product. E1, exon 1, E2, exon 2. Arrow indicates the position of the gRNA. ( g ) Immunofluorescence of AAVS1-EF1a-mTagRFP-T organoids. Blue: DAPI (nuclei); red: mTagRFP-T (membrane localised reporter); white: SOX2 (lung progenitor marker). Scale bars in all panels denote 50 μm.

Article Snippet: 6 × 10 5 cells were suspended using Lonza Nucleofection P3 buffer, mixed with 6 μg of SOX9 reporter repair template plasmid.

Techniques: Plasmid Preparation, Selection, Flow Cytometry, Microscopy, Immunofluorescence, Marker

Journal: eLife

Article Title: A functional genetic toolbox for human tissue-derived organoids

doi: 10.7554/eLife.67886

Figure Lengend Snippet: ( a ) Workflow for testing different homology-directed repair (HDR)-enhancing drugs. Organoid cells were treated with DMSO, RS-1, L755507 and SCR-7 for 48 hr after nucleofection for mEGFP-ACTB gene targeting. The percentage of EGFP-positive cells was analysed 72 hr after nucleofection. DMSO treatment was used as a negative control. ( b ) Summary of percentage of EGFP positive. No significant improvement of targeting efficiency was observed after the drug treatments. N = 3 different organoid lines were used. Error bars are plotted to show mean ± SEM.

Article Snippet: 6 × 10 5 cells were suspended using Lonza Nucleofection P3 buffer, mixed with 6 μg of SOX9 reporter repair template plasmid.

Techniques: Negative Control

Journal: Cell Genomics

Article Title: SpRY-mediated screens facilitate functional dissection of non-coding sequences at single-base resolution

doi: 10.1016/j.xgen.2024.100583

Figure Lengend Snippet:

Article Snippet: P3 Lonza buffer with supplement , Lonza , Cat#: V4XP-3032.

Techniques: Sequencing, Chromatin Immunoprecipitation, Binding Assay, Software, Recombinant, Modification, SYBR Green Assay, Purification, Sensitive Assay, Protease Inhibitor, Luciferase

Journal: The EMBO Journal

Article Title: CARD8 inflammasome activation triggers pyroptosis in human T cells

doi: 10.15252/embj.2020105071

Figure Lengend Snippet:

Article Snippet: P3 Primary Cell 96‐well Kit , Lonza , #V4SP‐3096.

Techniques: Sequencing, Synthesized, Enzyme-linked Immunosorbent Assay, Red Blood Cell Lysis, Cell Isolation, Sterility, Recombinant, CyQUANT Assay, LDH Cytotoxicity Assay, Staining, Bicinchoninic Acid Protein Assay, Protease Inhibitor, Western Blot, Membrane, Software