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Abstract
Methods to measure heterogeneity among cells are rapidly transforming our understanding of biology but are currently limited to molecular abundance measurements. We developed an approach to simultaneously measure biochemical activities and mRNA abundance in single cells to understand the heterogeneity of DNA repair activities across thousands of human lymphocytes, identifying known and novel cell-type-specific DNA repair phenotypes.
Highlights
We developed a functional assay as a new modality for single-cell experiments
The mRNA fraction was subjected to the standard protocol to measure gene expression for single cells, whereas the repair fraction was captured in a modified protocol (Supplementary Fig. 1)
We captured thousands of single cells with expected DNA repair defects: RNASEH2CKO cells could incise uracil but not a ribonucleotide repair substrate, and vice versa for UNGKO cells, with a few droplets containing more than one cell from each genotype and both uracil and ribonucleotide repair activities (Fig. 1c, d)
Summary
3. After GEM-RT cleanup, DNA repair substrates and products were separated from mRNA prior to cDNA amplification. The beads containing the RT products were washed twice with 150 μl of 80% ethanol, dried for 2 minutes at room temperature, and eluted in 35.5 μl of Elution Solution 1 according to 10x SPRIselect cleanup protocols. This fraction was used to prepare the mRNA expression library according to manufacturer's instructions. The beads were dried at room temperature for 2 minutes, eluted in 20 μl water This fraction was used to prepare the DNA repair libraries. The DNA repair libraries were prepared with the following steps: 1. End repair: 20 μl of the purified DNA repair libraries were added to an end repair reaction with a total volume of 30 μl (NEBNext End repair Module E6050) and incubated for 30 minutes at 20° C
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