Single-cell RNA sequencing is widely used in developmental biology, immunology, cancer research, and clinical applications, providing a scalable and reliable approach for single-cell transcriptomics. The Chromium Next GEM Single Cell 3′ Reagent kits by 10× Genomics provide an advanced method for generating single-cell gene expression libraries using microfluidic partitioning and barcoding technology. These kits enable the profiling of thousands of individual cells in a single experiment by encapsulating single cells with uniquely barcoded Gel Beads-in-Emulsion (GEMs). Reverse transcription (RT) occurs within each GEM, producing barcoded cDNA, which is subsequently purified, amplified, and converted into a dual-indexed sequencing library. The workflow consists of four major steps: GEM generation and barcoding, post-GEM-RT cleanup and cDNA amplification, 3′ gene expression library construction, and sequencing. Quality control measures, including SPRIselect bead cleanup, Bioanalyzer/TapeStation validation, and PCR optimization, ensure high-quality sequencing results. The final library is compatible with Illumina sequencing platforms, allowing researchers to analyze cellular heterogeneity, gene expression dynamics, and rare cell populations. This protocol is based on the 10× Chromium Single Cell 3′ Reagent Kits user guide (v3.1—Dual Index), which can be downloaded from the 10× Genomics website ( https://www.10xgenomics.com ). It is recommended to refer to the original official guide for more details when carrying out the experiments with these kits, ensuring you stay updated with any revisions or updates.