taps wash buffer  (Thermo Fisher)

Journal: bioRxiv

Article Title: Whole-genome single-cell multimodal history tracing to reveal cell identity transition

doi: 10.1101/2025.08.12.669973

Figure Lengend Snippet: a , Schematic of the droplet-based single-cell (sc) Dam&Tag protocol. One day after Dam-POI activation in the mouse cortex, the infected brain region is dissected, and nuclei are extracted. Permeabilized nuclei are incubated with V5-EGFP-m6A-Tracer, mouse anti-V5 antibody, and secondary anti-mouse IgG antibody. Subsequently, nuclei are tagmented by Protein A–Tn5 loaded with ME-A adapters bearing sample-specific barcodes. Following FACS-isolation of nuclei stained with V5-EGFP-m6A-Trace, nuclei are pooled and subjected to 10x Chromium scATAC-seq chemistry. Overloaded nuclei (i.e., multiple per droplet) receive cell-specific barcodes during the standard barcoding reaction. Dual indexing preserves both sample origin and single-cell identity for high-throughput m6A profiling in one Chromium channel. Following linear amplification, secondary tagmentation is carried out with the ME-B adapter. Library preparation PCR, and high-throughput sequencing are carried out. We use publicly available informatics tools for the downstream analysis. b , Representative FACS plots from adult mouse cortex expressing Dam-Leo1. 7-AAD signal is used to gate intact nuclei and single events are gated. Nuclei stained with V5-EGFP-m6A-Tracer show an EGFP+ population, which is gated to enrich m6A-marked neurons for downstream scDam&Tag library preparation. c , UMAP embedding per replicate with cell type-specific genes.

Article Snippet: For library preparation using in-house Tn5 transposase (12.5 μM, loaded with annealed adapters, Supplementary Table), 5 μL of 0.2 ng/μL purified cDNA was mixed with 15 μL tagmentation buffer (prepared using 5× TAPS-DMF buffer containing 50 mM TAPS pH 8.5 (Thermofisher, J63268.AE), 25 mM MgCl2, and 50% Dimethylformamide (VWR, TCIAD0722)), incubated at 55 °C for 7 min, and the reaction was terminated with 5 μL of 0.2% SDS.

Techniques: Activation Assay, Infection, Incubation, Isolation, Staining, High Throughput Screening Assay, Amplification, Next-Generation Sequencing, Expressing

Journal: bioRxiv

Article Title: Whole-genome single-cell multimodal history tracing to reveal cell identity transition

doi: 10.1101/2025.08.12.669973

Figure Lengend Snippet: a , Schematic of single-cell (sc) HisTrac-seq for simultaneous profiling of past and present molecular profiles. After Dam-POI activation, nuclei are extracted and permeabilized. Permeabilized nuclei are first treated with V5-EGFP-m6A-Tracer to bind m6A marks. Mouse anti-V5 antibody and anti-mouse-IgG nanobody–Tn5 tagment chromatin at historical (m6A) marks. In parallel, rabbit anti-H3K27ac antibody and anti-rabbit-IgG nanobody–Tn5 target active histone modifications to profile present transcriptional activity. ME-A adapters carry modality- and sample-specific barcodes, enabling post-sequencing demultiplexing of m6A versus H3K27ac modalities and sample information. Following FACS-isolation of nuclei stained with V5-EGFP-m6A-Tracer, nuclei are pooled and subjected to 10x Chromium scATAC-seq chemistry. Overloaded nuclei (i.e., multiple per droplet) receive cell-specific barcodes during the standard barcoding reaction. Dual indexing preserves sample origin, modality, and single-cell identity for high-throughput m6A profiling in one Chromium channel. Following linear amplification, secondary tagmentation is carried out with the ME-B adapter. Library preparation PCR and high-throughput sequencing are carried out. We use publicly available informatics tools for the downstream analysis. b , Representative FACS plots from mESC-derived neurons expressing Dam-Leo1. Nuclei stained with V5-EGFP-m6A-Tracer show an EGFP+ population, which is gated to enrich m6A-marked neurons for downstream library preparation. 7-AAD signal is used to gate intact nuclei.

Article Snippet: For library preparation using in-house Tn5 transposase (12.5 μM, loaded with annealed adapters, Supplementary Table), 5 μL of 0.2 ng/μL purified cDNA was mixed with 15 μL tagmentation buffer (prepared using 5× TAPS-DMF buffer containing 50 mM TAPS pH 8.5 (Thermofisher, J63268.AE), 25 mM MgCl2, and 50% Dimethylformamide (VWR, TCIAD0722)), incubated at 55 °C for 7 min, and the reaction was terminated with 5 μL of 0.2% SDS.

Techniques: Activation Assay, Activity Assay, Sequencing, Isolation, Staining, High Throughput Screening Assay, Amplification, Next-Generation Sequencing, Derivative Assay, Expressing