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Harnessing HetHydrogel: An Approach to Single-Cell Multi-Omics

ABSTRACT: Developing a single-cell multi-omics technology with mtDNA mutation profiling at its core will enable precise determination of the relationship between mtDNA mutations and cellular phenotypes. To date, the development of permeability-engineered compartmentalization for the isolation and single-cell library preparation of individual cells, which includes Tn5 tagmentation and single-cell barcoding operations in the permeabilized microcapsule format, remains a formidable challenge. Herein, we present a approach for single-cell mitochondrial analysis, harnessing microfluidics to generate heterogeneous hydrogels as artificial membranes. These hydrogels serve to regulate the passage of mitochondrial DNA and nuclear genomes subsequent to the cell lysis step. This approach enables high-throughput mitochondrial DNA genotyping and accessible chromatin profiling at the single-cell level, significantly enhancing the capabilities of permeability-engineered compartmentalization.We conducted a comprehensive evaluation of cell retention and the selective permeability of the capsule reaction system, optimizing conditions for cell lysis, Tn5 tagmentation, and barcode labeling. Validation experiments were performed using Human 293T and Mouse 3T3 cells to assess identification and quantification performance. Our method offers a new strategy for the study of the functions of various mtDNA mutations in biological processes, while also offering a new system for the construction of single-cell multi-omics libraries.

ORGANISM(S): Mus musculus Homo sapiens

PROVIDER: GSE244725 | GEO | 2023/10/10

REPOSITORIES: GEO

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Project description:Smart-seq3xpress was carefully optimized and >1,000 conditions were evaluated. This data submission is organized in 15 datasets that each contain fastq files, unmapped bam files, read count tables, UMI count tables and a barcode annotation file. The barcode_annotation.txt files contain the exact factors/variables tested. Below a short description of each set of experiments: K562_lowvolume: Evaluation of scaling volumes of Smart-seq3 (indicated volume refers to total volume in PCR), whether overlay was used and if cDNA was bead-cleaned or diluted prior to tagmentation. Cell input was K562 cells. The columns \\"treatment\\", \\"volume\\" and \\"VL\\" indicate the experimental parameters. HEK_lowvolume: Evaluation of scaling volumes of Smart-seq3 (indicated volume refers to total volume in PCR), whether overlay was used and if cDNA was bead-cleaned or diluted prior to tagmentation. Cell input was HEK293FT cells. The columns \\"treatment\\", \\"volume\\" and \\"VL\\" indicate the experimental parameters. overlays: Evaluation of the effect of various overlays when generating HEK293FT libraries in 1 uL total volume. The column \\"condition\\" indicates the applied overlay. tagmentations: Evaluation of input cDNA vs Tn5 amount during tagmentation. Purified cDNA from one 384-well plate was used as input into various conditions of tagmentations. The experiments contain evaluation of cDNA amount with fixed Tn5 amount or varying Tn5 amount while keeping the default volume (2 uL) of the tagmentation reaction or scaling the reaction volume. The column \\"condition\\" contains a string indicating reaction volume, cDNA input and Tn5 ATM enzyme amount. If no volume is indicated, reaction was performed in 2 uL. HomeTn5: Evaluation of tagmentation using in-house produced Tn5 enzyme (Picelli et al., 2015) when tagmenting cDNA generated from HEK293FT cells in 1 uL total volume. The column \\"Tn5concentration\\" indicates the Tn5 reaction concentration at 2 uL reaction volume. cycles_cleanups: Optimization of Smart-seq3xpress (column \\"experiment\\" shows \\"direct_tag\\") in regards to clean-ups after cDNA synthesis (column \\"condition\\": noclean, Exo+FastAP, ExoSAP) and dilution volume (9 or 19 uL); PCR cycle numbers (column \\"pcr_input\\") and ATM Tn5 enzyme amount (column \\"ATM\\"). Cell input was HEK293FT cells. PreAmp_Polymerase: Evaluation of various PCR polymerases during initial cDNA amplification. The polymerases are indicated in column \\"polymerase\\". We also evaluated several TSO concentrations (concentration in RT is given) and fwd/rev PCR primers (concentration given in PCR reaction). Cell input was HEK293FT cells. TDE1: Large optimization of tagmentation conditions using the TDE1 Tn5 enzyme. We varied reactions by changing PCR polymerase (KAPA / SeqAmp), PCR extension time and the number of PCR cycles during cDNA amplification. During tagmentation, we varied the amount of TDE1 enzyme, the amount of DMF in the tagmentation reaction buffer and the presence of Tween-20 in the final post-tagmentation PCR. Cell input was HEK293FT cells. TSOs_RT_v1-7: Large scale evaluation of conditions relating to RT and PCR, with a focus on new template-switching oligo (TSO) designs. In total, >20,000 cells and >500 conditions are contained in these datasets. The barcode annotation file contains precise information on the reaction conditions of Lysis, RT, PCR as well as utilized TSO designs. Data was generated from HEK293FT cells and hPBMC (Lonza).

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Harnessing HetHydrogel: An Approach to Single-Cell Multi-Omics

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