TY - JOUR
T1 - lentiMPRA and MPRAflow for high-throughput functional characterization of gene regulatory elements
AU - Gordon, M. Grace
AU - Inoue, Fumitaka
AU - Martin, Beth
AU - Schubach, Max
AU - Agarwal, Vikram
AU - Whalen, Sean
AU - Feng, Shiyun
AU - Zhao, Jingjing
AU - Ashuach, Tal
AU - Ziffra, Ryan
AU - Kreimer, Anat
AU - Georgakopoulous-Soares, Ilias
AU - Yosef, Nir
AU - Ye, Chun Jimmie
AU - Pollard, Katherine S.
AU - Shendure, Jay
AU - Kircher, Martin
AU - Ahituv, Nadav
N1 - Publisher Copyright:
© 2020, The Author(s), under exclusive licence to Springer Nature Limited.
PY - 2020/8/1
Y1 - 2020/8/1
N2 - Massively parallel reporter assays (MPRAs) can simultaneously measure the function of thousands of candidate regulatory sequences (CRSs) in a quantitative manner. In this method, CRSs are cloned upstream of a minimal promoter and reporter gene, alongside a unique barcode, and introduced into cells. If the CRS is a functional regulatory element, it will lead to the transcription of the barcode sequence, which is measured via RNA sequencing and normalized for cellular integration via DNA sequencing of the barcode. This technology has been used to test thousands of sequences and their variants for regulatory activity, to decipher the regulatory code and its evolution, and to develop genetic switches. Lentivirus-based MPRA (lentiMPRA) produces ‘in-genome’ readouts and enables the use of this technique in hard-to-transfect cells. Here, we provide a detailed protocol for lentiMPRA, along with a user-friendly Nextflow-based computational pipeline—MPRAflow—for quantifying CRS activity from different MPRA designs. The lentiMPRA protocol takes ~2 months, which includes sequencing turnaround time and data processing with MPRAflow.
AB - Massively parallel reporter assays (MPRAs) can simultaneously measure the function of thousands of candidate regulatory sequences (CRSs) in a quantitative manner. In this method, CRSs are cloned upstream of a minimal promoter and reporter gene, alongside a unique barcode, and introduced into cells. If the CRS is a functional regulatory element, it will lead to the transcription of the barcode sequence, which is measured via RNA sequencing and normalized for cellular integration via DNA sequencing of the barcode. This technology has been used to test thousands of sequences and their variants for regulatory activity, to decipher the regulatory code and its evolution, and to develop genetic switches. Lentivirus-based MPRA (lentiMPRA) produces ‘in-genome’ readouts and enables the use of this technique in hard-to-transfect cells. Here, we provide a detailed protocol for lentiMPRA, along with a user-friendly Nextflow-based computational pipeline—MPRAflow—for quantifying CRS activity from different MPRA designs. The lentiMPRA protocol takes ~2 months, which includes sequencing turnaround time and data processing with MPRAflow.
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U2 - 10.1038/s41596-020-0333-5
DO - 10.1038/s41596-020-0333-5
M3 - Article
C2 - 32641802
AN - SCOPUS:85087706837
SN - 1754-2189
VL - 15
SP - 2387
EP - 2412
JO - Nature Protocols
JF - Nature Protocols
IS - 8
ER -