Single Cell & Spatial Omics Core

10X Chromium Next GEM Single Cell 3’ Gene Expression Workflow

Chromium Next GEM Chip:

• Up to 8 samples per chip processed in parallel
• 500 to 10,000 cells per channel
• 18 minutes run time per chip
• Preferred cell size < 30 µm
• ~60-65 % cell recovery efficiency

https://www.youtube.com/watch?v=PAiI1rarAPk

Single-cell RNA sequencing (scRNA-Seq) library preparation, which utilizes a microfluidics-free workflow based on PIPseq technology. These kits are designed for accessibility, scalability, and high-quality data generation.

The kit captures the 3' end of mRNA transcripts and are compatible with various sample types including blood, bone marrow, and single cells from human, mouse, yeast, and zebrafish ranging from 2,000 – 100,000 cells per sample.

It is a simple, manual workflow with flexible stopping points that can be performed on a benchtop, eliminating the need for expensive microfluidic equipment. The process involves cell capture and barcoding using particle-templated instant partitions (PIPs), followed by cDNA generation and library preparation for sequencing.

Figure 1. ILMN single cell preparation: Monodisperse emulsion by vortexing

1. Cell Capture and Lysis

• Mixing: Single-cell or nuclei suspensions are mixed with barcoded hydrogel template particles (PIPs) and oil.
• Emulsification: Using an engineered vortex mixer, the mixture is emulsified to create millions of partitions, each potentially containing one cell and one barcoded bead.
• Lysis: Cells are lysed within the partitions to release mRNA, which then binds to the poly(dT) primers on the barcoded beads. 

2. cDNA Synthesis

• Emulsion Breaking: The emulsion is chemically broken and washed to recover the beads.
• Reverse Transcription: Beads undergo bulk reverse transcription to generate full-length cDNA containing unique cell barcodes and intrinsic molecular identifiers (IMIs). 

3. Library Preparation

• Fragmentation & Tagging: The cDNA is fragmented, and adapters are ligated for sequencing compatibility.
• Amplification: The library is amplified via PCR to ensure sufficient concentration for sequencing. 

4. Sequencing and Analysis

• Sequencing: Libraries are sequenced on compatible Illumina platforms (e.g., NextSeq 2000, NovaSeq X) using paired-end reads.
• Data Analysis: Resulting data is processed via DRAGEN secondary analysis software for cell calling and gene expression profiling. 

• Time: ~15 hours total (10 minutes for cell capture).
• Scalability: Supports 100 to 200,000+ cells per sample.
• Compatibility: Works with fresh or fixed cells (DSP / Methanol fixation).
• Equipment: Requires the Illumina Single Cell Prep Starter Equipment, which includes a specific vortex mixer and dry bath for precise emulsion handling. 

Figure 2. Single cell mRNA capture and barcoding with PIP-Seq chemistry

scRNA-Seq for cryopreserved human peripheral blood mononuclear cells (PBMCs). UMAP of 31,613 cells captured using the Illumina Single Cell 3ʹ RNA Prep T20 kit resolves diverse immune cell populations.

Figure 3. High-resolution scRNA-Seq at a scale of tens of thousands of cells

scRNA-Seq for mouse brain nuclei from frozen tissue. UMAP analysis of 155,000 nuclei captured using the T100 kit resolves diverse brain cell populations.

Figure 4. High-resolution scRNA-Seq at a scale of hundreds of thousands of cells