Microwell-seq uses a special agarose microplate, the Microwell, as a platform for single cell capture. It has the characteristics of high throughput, low cost, and high sequencing quality. Microwell adds single cells and beads to the wells for reaction, ensuring that the signals of each well will not interfere with each other, so as to accurately label the cellular source of RNA.
Overview
Microwell is made of a silicon wafer engraved with about 100,000 small wells as a carrier, and then wrapped with PDMS and Agarose on it. After the single cell is loaded into the Microwell, it will fall into the small well of the wafer, and a small well can only accommodate a single cell and one magnetic bead. Each small well is equivalent to an independent reaction chamber, where subsequent cell lysis and magnetic bead adsorption operations will be completed. This ensures that the RNA on a magnetic bead originates from only one cell. The advantages of Microwell-Seq include: (1) the silicon wafers that capture single cells can be reused to save the cost of equipment; (2) the cell throughput is above 5k, which is higher than that of the FACS method; (3) The library construction cost is low; (4) Microscopic quality control is added in the process of cell sorting to improve the quality of single cell sorting.
Using Microwell-Seq technology, CD Genomics can perform single-cell transcriptome analysis on thousands of cells at the same time, and use magnetic bead labeling technology to add specific tags to single cells. cDNA library is synthesized by reverse transcription and followed by amplification. The obtained cDNA can be used for subsequent sequencing studies. Drop-Seq can be applied to fields such as tumor heterogeneity，immunocyte and immune mechanism，stem cells，embryology, etc.
Features
High-throughput and Efficient
High-throughput at Low Cost
Single-Cell Insights
Multiple Applications
~10,000 cells per chip, parallel analysis of multiple chips; > 90% mRNA coverage.
High throughput sequencing in a short time; Minimized consumption of expensive samples.
Analyze sequences of single-cells in a highly parallel manner.
Understanding complex tissues, tumor heterogeneity and clonal evolution.
Project Workflow
1. Sample Preparation
Load cell suspension, pick out cell doublets, load bead suspension, wash beads, and image.
2. Library Preparation
From reverse transcription and exonuclease I treatment to cDNA library preparation.
3. Sequencing Platform
Illumina HiSeq; PE50/75/100/150; >10G clean data
4. Data Analysis
Visualize and preprocess results, and perform custom bioinformatics analysis.
Bioinformatics Analysis Pipeline
In-depth data analysis:
Raw data QC and clean-up
Estimation of sequencing depth and coverage
Differentially expressed gene analysis
SNP/InDel/CNV/SV calling
GO and KEGG enrichment analysis
Gene interaction network
Alternative pre-mRNA splicing
Detection of RNA editing, novel transcripts, and fusion genes
Digital Gene Expression
Sample Requirements
RNA amount: Total RNA ≥ 5 ug (without degradation or DNA contamination); RNA purity: OD260/280 = 1.8~2.2; OD260/230 ≥ 1.5; RNA quality: 28S:18S ≥ 1.5，RIN ≥ 7
Please make sure that the RNA is not significantly degraded.
Sample storage: RNA can be dissolved in ethanol or RNA-free ultra-pure water and stored at -80°C. RNA should avoid repeated freezing and thawing.
Shipping Method:When shipping RNA samples, the RNA sample is stored in a 1.5 mL Eppendorf tube, sealed with sealing film. Shipments are generally recommended to contain 5-10 pounds of dry ice per 24 hours.
