This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Method Article
A protocol for Targeted Perturb (TAP)-seq and targeted single-cell RNA-seq
Lars M. Steinmetz
European Molecular Biology Laboratory (EMBL), Genome Biology Unit, 69117 Heidelberg, Germany; Department of Genetics, Stanford University School of Medicine, Stanford, California 94305, USA; Stanford Genome Technology Center, Palo Alto, California 94304, USA
Corresponding Author
ORCiD: https://orcid.org/0000-0002-3962-2865
License:
This work is licensed under a CC BY 4.0 License. Read Full License
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.
Keywords
single-cell, CRISPR, Cas9, CRISPRi, functional genomics, genetic screens, RNA sequencing
Figure 1
Figure 2
Figure 3
Lars Velten
ORCiDcommented on 15 September, 2020
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.
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
History
CURRENT STATUS: Posted
Version 1
Posted 02 Jun, 2020
Community comments: 1
Metrics
Comments: 1
HTML Views: ...
Associated Publications
Learn more about our company.
This is a preprint. It has not completed peer review.
Method Article
A protocol for Targeted Perturb (TAP)-seq and targeted single-cell RNA-seq
Lars M. Steinmetz
European Molecular Biology Laboratory (EMBL), Genome Biology Unit, 69117 Heidelberg, Germany; Department of Genetics, Stanford University School of Medicine, Stanford, California 94305, USA; Stanford Genome Technology Center, Palo Alto, California 94304, USA
Corresponding Author
ORCiD: https://orcid.org/0000-0002-3962-2865
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
History
CURRENT STATUS: Posted
Version 1
Posted 02 Jun, 2020
Community comments: 1
Metrics
Comments: 1
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
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
Lars Velten
ORCiDcommented on 15 September, 2020
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.