Question: Can I pool 3’ and 5’ Single Cell Gene Expression libraries for use in the Targeted Gene Expression assay?
Answer: For the Targeted Gene Expression assay, up to eight Whole Transcriptome Analysis (WTA) libraries can be pooled prior to hybridization. For optimal performance, we recommend pooling WTA libraries of the same assay type. If 3’ and 5’ Single Cell Gene Expression (scGEX) libraries are pooled, this can lead to unexpected read balance coming off the sequencer, with a shift in read distribution in favor of 5' libraries.
During in-house tests, we have found that targeted libraries prepared from 5’ scGEX libraries tend to have higher yields than targeted libraries prepared from 3’ scGEX libraries. This is hypothesized to be due to differences in the typical melting temperatures of 5’ versus 3’ ends of transcripts.
When targeted libraries are generated from pooled 5’ and 3’ scGEX libraries, we often observe a shift in read distribution in favor of 5’ scGEX libraries. This is likely due to the underlying differences in targeted library yield between the two input library types.
The specificity and sensitivity of the targeted assay are typically unaffected by pooling 5’ and 3’ libraries. The main consequence is expected to be a shift in the final read distribution.
Products: Targeted Gene Expression