Question: Why were the lysis conditions updated in protocol CG000124 for Isolation of Nuclei for Single Cell RNA Sequencing?
Answer: To improve assay performance, protocol CG000124 was updated from RevD to RevE in March 2021.
When testing our previous protocol (RevD) on additional sample types, we found that some cell types were being over-lysed. We, therefore, performed optimization experiments to identify a new lysis protocol that allows for consistent performance across a range of sample types. The specific changes are outlined below.
1) Protocol for isolating nuclei from single-cell suspensions
The new protocol (RevE) has the following updates:
- A lower volume of Lysis Buffer (200 µl instead of 1 ml)
- Shorter lysis time (1 min instead of 5 min)
- Lower detergent concentration in Lysis Buffer (0.025% NP40 instead of 0.1%)
- Using PBS as Lysis Buffer diluent
- Increased centrifugation time post-lysis (10 min instead of 5 min)
2) Protocol for isolating nuclei from embryonic mouse brain tissue
The new protocol (RevE) has the following updates:
- A lower volume of Lysis Buffer (400 µl instead of 2 ml)
- Shorter lysis time (10 min instead of 15 min)
- Increased centrifugation time post-lysis (10 min instead of 5 min)
Please note that, when working with a new sample type, we still recommend performing pilot experiments to find the optimal lysis time for your sample. See: What are the best practices for working with nuclei samples for 3' single-cell gene expression?
Over-lysis can reduce data quality by leading to an increase in the background (i.e., decreased “Fraction Reads in Cells”) and lower library complexity (i.e., lower Genes/UMIs per cell).
Products: Single Cell Gene Expression, CellPlex