# Protocol update to: High-throughput scNMT protocol for multiomics profiling of single cells from mouse brain and pancreatic organoids

## TL;DR

This paper updates a protocol for high-throughput single-cell multiomics profiling, improving efficiency and data quality in mouse brain and pancreatic organoid studies.

## Contribution

The protocol is updated to a 384-well format with reduced costs and improved cDNA quality using Smart-seq3 and patterned-flow cells.

## Key findings

- Throughput increased to 384-well format with reduced costs.
- Improved detection of genes and CpGs per cell using Smart-seq3.
- Protocol validated in two research papers with enhanced troubleshooting.

## Abstract

Single-cell nucleosome, methylome, and transcriptome (scNMT) sequencing is a recently developed method that allows multiomics profiling of single cells. In this scNMT protocol, we describe profiling of cells from mouse brain and pancreatic organoids, using liquid handling platforms to increase throughput from 96-well to 384-well plate format. Our approach miniaturizes reaction volumes and incorporates the latest Smart-seq3 protocol to obtain higher numbers of detected genes and genomic DNA (gDNA) CpGs per cell. We outline normalization steps to optimally distribute per-cell sequencing depth.

For complete details on the use and execution of this protocol, please refer to Kremer et al. and other works.1,2,3,4,5,6,7

This protocol is an update to Cerrizuela et al.7

•Throughput increase to 384-well plate format for methylome with a decrease in costs•Validation of the protocol in two research papers•Improved troubleshooting for optimization of cDNA quality•Incorporation of patterned-flow cells for sequencing of transcriptomic libraries

Throughput increase to 384-well plate format for methylome with a decrease in costs

Validation of the protocol in two research papers

Improved troubleshooting for optimization of cDNA quality

Incorporation of patterned-flow cells for sequencing of transcriptomic libraries

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Single-cell nucleosome, methylome, and transcriptome (scNMT) sequencing is a recently developed method that allows multiomics profiling of single cells. In this scNMT protocol, we describe profiling of cells from mouse brain and pancreatic organoids, using liquid handling platforms to increase throughput from 96-well to 384-well plate format. Our approach miniaturizes reaction volumes and incorporates the latest Smart-seq3 protocol to obtain higher numbers of detected genes and genomic DNA (gDNA) CpGs per cell. We outline normalization steps to optimally distribute per-cell sequencing depth.