Laminar Wash Sample Preparation for Single-Cell Sequencing
Complete cell processing in five minutes.
Single-cell genomics is rapidly growing. Nature Methods named multimodal omics the method of the year for 2019. And as more scientists use single-cell techniques, they find that sample preparation is very important to achieve high-quality data.
Single cell sequencing can suffer from poor data quality due to clumping of cells, incomplete washing of cells leaving cell-free nucleic acids, and poor retention of low numbers of cells. Washing via centrifugation causes stress on the cells in samples and can cause erroneous gene expression results.
Sample prep is extremely important in single-cell genomics, especially for single cell gene expression and immune profiling. In this area, mRNA transcripts are being barcoded and cells can experience mRNA leakage (causing increased background) or mRNA degradation (causing less signal). Unlike with flow cytometry, in single-cell sequencing it is not possible to gate out debris or groups of cells. The cell preparation must be as clean as possible and consist of single cells.
10X Genomics’ Sample Preparation Demonstrated Protocol Fresh Frozen Human Peripheral Blood Mononuclear Cells for Single Cell RNA Sequencing (Part Number CG00039 Rev C) uses centrifugation to wash cells and warns “However, be careful not to over-centrifuge as increased centrifugation speed can compromise the cell integrity and viability.” So, it makes sense that if there is a gentler method to wash cells scientists should use it.
By using the Laminar Wash system scientists can see time savings and less manual manipulation.
Laminar Wash systems can wash cells without putting stress on cells that may impact gene expression, cell integrity and viability.
- No need to spin cells to concentrate them
- Reduction of cell debris
- Minimize cell clumping
Nuclei Isolation using Laminar Wash
PBMCs were lysed in bulk in tube with Lysis Buffer, quenched with Wash Buffer and centrifuged
Cells are further either washed twiced in tube by centrifuge, OR plated in Laminar Wash plate to settle for 20min, washed 7x in a Laminar Wash HT1000 System with PBS + 0.04% BSA.
Centrifuged samples have inconsistent retention, with examples of large clumps. Nuclei are more distinct when washed on Laminar Wash systems. Retention numbers with Laminar Wash systems are higher and more consistent.
Efficient washing of IL-7 in T-cell stimulation kinetics assay
Complete washing is critical in single cell genomics assays. The figure below illustrates an assay to investigate kinetics of IL-7 signal decay by quantification of Stat5 phosphorylation after IL-7 removal. The challenge is, T cells are extremely sensitive to minute amounts of IL-7. Centrifugation wash takes a long time (3 rounds for complete dilution), stress on cells notwithstanding. Laminar Wash HT1000 System shows efficient removal of IL-7 in less than 5 minutes (18x wash), shown by faster decay of pStat5 after IL-7 removal.