Introduction
Single cell RNA sequencing (scRNA seq) is a technique used for unbiased characterization of gene expression in complex cell and tissue samples. Although scRNA seq chedi has changed modern biology, it remains technically challenging and often costly. The current solutions on the market can effectively analyze a small subset of cells in an initial pool with tens of thousands of cells. However, a simplified approach for preparing scRNA seq libraries for rare cell populations is still lacking. Here, we have developed a novel well plate detection method for cost-effective and highly sensitive scRNA seq. This workflow supports the preparation of rare sample libraries from rare cell populations. In this new workflow (Figure 1), we used DispenCell and a dedicated detection pipette (SEED Biosciences, Switzerland), along with a specialized and optimized single-cell library preparation system (MERCURIUS high-sensitivity BRB seq kit, Alithea Genomics， Switzerland).
Single cell allocation
Firstly, before preparing the sequencing library, we tested the ability of DispenCell to allocate single cells in a dedicated lysis buffer. The lymphocyte lines were thawed from frozen vials, passaged twice, and then harvested in single-cell suspension. Filter cells using a 10 μ m cell filter (Milton Biotec, Germany), count and dilute to a final concentration of 2 × 104 cells/mL in RNAse free methylcellulose distribution solution (DispenMe, SEED Biosciences). Then, 200 cells were loaded from the cell suspension using DispenCell sensing tips. Allocate single cells into 384 well plates using DispenCell, pre loaded with 4.2 μ L lysis buffer (HS BRB seq kit, Alicea Genomics). The first 96 holes are allocated for approximately 7 minutes. Then, stop the allocation because we have enough samples for downstream analysis.
Single cell quality control
After allocation, we immediately used the software DispenSoft developed by SEED Biosciences for single-cell quality control post-processing. Each hole of the orifice plate is represented by a color coded matrix (Figure 2a). When a hole contains a single cell, the software marks it as green, while a red hole contains zero or more cells and should be discarded. To distinguish between fragments and cells, a threshold was set by gating the size based histogram (Figure 2b). Only peaks above the threshold are considered cells.
Here, we obtained 77 single-cell wells (80% single-cell isolation rate). Using DispenSoft, the raw impedance data can also be used to record the process. Each impedance curve can be manually checked. A single peak is a characteristic of a single cell (Figure 2. c), while multiple cells produce multiple peaks. This indicates that DispenCell can allocate single cells in lysis buffer in a simple, fast, and traceable process.