A protocol for Targeted Perturb (TAP)-seq and targeted single-cell RNA-seq
Here we provide a step-by-step protocol for targeted single-cell RNA-seq and targeted Perturb-seq, as reported in the linked publication Schraivogel et al. Nat Meth 2020. The protocol describes cell preparation using flow cytometry, single-cell droplet formation with 10X Genomics, and targeted 3’ single-cell RNA-seq library preparation for Illumina sequencing.
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We have received a couple of requests to combine TAP-seq with whole transcriptome sequencing. In our experience what works is to follow the protocol of your single cell platform and retain some amplified, non-tagmented cDNA. For example, in the case of 10x genomics, we retain an aliquot of the cDNA obtained from step 2.3 ("cDNA cleanup") of their protocol and otherwise complete the protocol like normal to obtain a whole transcriptome library. 10ng of cDNA then serve as input into step 16 (PCR 1) of this TAP-seq protocol, which is performed with a small number of cycles (10-11). Subsequently we follow the TAP-seq protocol to obtain a targeted library. The two libraries are then pooled for sequencing at the desired ratio. The quantitative performance of this modified protocol might be lower due to the additional PCR cycles, and we do not yet have sufficient data to comment on its quantitative performance. However it appears to be convenient to increase the sensitivity of detection for specific genes of interest.
Posted 02 Jun, 2020
A protocol for Targeted Perturb (TAP)-seq and targeted single-cell RNA-seq
Posted 02 Jun, 2020
Here we provide a step-by-step protocol for targeted single-cell RNA-seq and targeted Perturb-seq, as reported in the linked publication Schraivogel et al. Nat Meth 2020. The protocol describes cell preparation using flow cytometry, single-cell droplet formation with 10X Genomics, and targeted 3’ single-cell RNA-seq library preparation for Illumina sequencing.
Figure 1
Figure 2
Figure 3
We have received a couple of requests to combine TAP-seq with whole transcriptome sequencing. In our experience what works is to follow the protocol of your single cell platform and retain some amplified, non-tagmented cDNA. For example, in the case of 10x genomics, we retain an aliquot of the cDNA obtained from step 2.3 ("cDNA cleanup") of their protocol and otherwise complete the protocol like normal to obtain a whole transcriptome library. 10ng of cDNA then serve as input into step 16 (PCR 1) of this TAP-seq protocol, which is performed with a small number of cycles (10-11). Subsequently we follow the TAP-seq protocol to obtain a targeted library. The two libraries are then pooled for sequencing at the desired ratio. The quantitative performance of this modified protocol might be lower due to the additional PCR cycles, and we do not yet have sufficient data to comment on its quantitative performance. However it appears to be convenient to increase the sensitivity of detection for specific genes of interest.
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