# Protocol to identify defined reprogramming factor expression using a factor-indexing single-nuclei multiome sequencing approach

## TL;DR

This paper introduces a protocol using a new sequencing method to track specific gene activity during cell reprogramming.

## Contribution

A novel factor-indexing single-nuclei multiome sequencing approach for tracking reprogramming factors.

## Key findings

- Barcoding transcription factors allows tracking of their expression in reprogramming cells.
- Human fibroblasts can be reprogrammed into pancreatic ductal-like cells using defined factors.
- FI-snMultiome-seq enables analysis of heterogeneous reprogramming cell populations.

## Abstract

Ectopic expression of lineage-specific transcription factors (TFs) of another cell type can induce cell fate reprogramming. However, the heterogeneity of reprogramming cells has been a challenge for data interpretation and model evaluation. Here, we present a protocol to characterize cells expressing defined factors during direct cell reprogramming using a factor-indexing approach based on single-nuclei multiome sequencing (FI-snMultiome-seq). We describe the steps for barcoding TFs, converting human fibroblasts to pancreatic ductal-like cells using defined TFs, and preparing library for FI-snMultiome-seq analysis.

For complete details on the use and execution of this protocol, please refer to Fei et al.1

•Generating barcoded Gateway destination vectors by PCR•Barcoding individual transcription factor constructs by Gateway cloning•Direct reprogramming of pancreatic ductal-like cells from human fibroblasts•Characterizing the cells expressing defined TFs in a heterogeneous cell population

Generating barcoded Gateway destination vectors by PCR

Barcoding individual transcription factor constructs by Gateway cloning

Direct reprogramming of pancreatic ductal-like cells from human fibroblasts

Characterizing the cells expressing defined TFs in a heterogeneous cell population

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

Ectopic expression of lineage-specific transcription factors (TFs) of another cell type can induce cell fate reprogramming. However, the heterogeneity of reprogramming cells has been a challenge for data interpretation and model evaluation. Here, we present a protocol to characterize cells expressing defined factors during direct cell reprogramming using a factor-indexing approach based on single-nuclei multiome sequencing (FI-snMultiome-seq). We describe the steps for barcoding TFs, converting human fibroblasts to pancreatic ductal-like cells using defined TFs, and preparing library for FI-snMultiome-seq analysis.