PMID- 32641802 OWN - NLM STAT- MEDLINE DCOM- 20200914 LR - 20240329 IS - 1750-2799 (Electronic) IS - 1754-2189 (Print) IS - 1750-2799 (Linking) VI - 15 IP - 8 DP - 2020 Aug TI - lentiMPRA and MPRAflow for high-throughput functional characterization of gene regulatory elements. PG - 2387-2412 LID - 10.1038/s41596-020-0333-5 [doi] 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. FAU - Gordon, M Grace AU - Gordon MG AUID- ORCID: 0000-0003-4321-9187 AD - Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, San Francisco, CA, USA. AD - Institute for Human Genetics, University of California, San Francisco, San Francisco, CA, USA. AD - Biological and Medical Informatics Graduate Program, University of California, San Francisco, San Francisco, CA, USA. FAU - Inoue, Fumitaka AU - Inoue F AUID- ORCID: 0000-0003-0657-434X AD - Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, San Francisco, CA, USA. fumitaka.inoue@ucsf.edu. AD - Institute for Human Genetics, University of California, San Francisco, San Francisco, CA, USA. fumitaka.inoue@ucsf.edu. FAU - Martin, Beth AU - Martin B AD - Department of Genome Sciences, University of Washington, Seattle, WA, USA. FAU - Schubach, Max AU - Schubach M AUID- ORCID: 0000-0002-2032-6679 AD - Berlin Institute of Health (BIH), Berlin, Germany. AD - Charite-Universitatsmedizin Berlin, Berlin, Germany. FAU - Agarwal, Vikram AU - Agarwal V AD - Department of Genome Sciences, University of Washington, Seattle, WA, USA. AD - Calico Life Sciences LLC, South San Francisco, CA, USA. FAU - Whalen, Sean AU - Whalen S AD - Gladstone Institutes, San Francisco, CA, USA. FAU - Feng, Shiyun AU - Feng S AD - Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, San Francisco, CA, USA. AD - Institute for Human Genetics, University of California, San Francisco, San Francisco, CA, USA. FAU - Zhao, Jingjing AU - Zhao J AD - Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, San Francisco, CA, USA. AD - Institute for Human Genetics, University of California, San Francisco, San Francisco, CA, USA. FAU - Ashuach, Tal AU - Ashuach T AD - Department of Electrical Engineering and Computer Sciences and Center for Computational Biology, University of California, Berkeley, Berkeley, CA, USA. FAU - Ziffra, Ryan AU - Ziffra R AD - Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, San Francisco, CA, USA. AD - Institute for Human Genetics, University of California, San Francisco, San Francisco, CA, USA. FAU - Kreimer, Anat AU - Kreimer A AD - Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, San Francisco, CA, USA. AD - Institute for Human Genetics, University of California, San Francisco, San Francisco, CA, USA. AD - Department of Electrical Engineering and Computer Sciences and Center for Computational Biology, University of California, Berkeley, Berkeley, CA, USA. FAU - Georgakopoulos-Soares, Ilias AU - Georgakopoulos-Soares I AD - Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, San Francisco, CA, USA. AD - Institute for Human Genetics, University of California, San Francisco, San Francisco, CA, USA. FAU - Yosef, Nir AU - Yosef N AD - Department of Electrical Engineering and Computer Sciences and Center for Computational Biology, University of California, Berkeley, Berkeley, CA, USA. AD - Chan-Zuckerberg Biohub, San Francisco, CA, USA. FAU - Ye, Chun Jimmie AU - Ye CJ AUID- ORCID: 0000-0001-6560-3783 AD - Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, San Francisco, CA, USA. AD - Institute for Human Genetics, University of California, San Francisco, San Francisco, CA, USA. AD - Chan-Zuckerberg Biohub, San Francisco, CA, USA. AD - Division of Rheumatology, Department of Medicine, University of California, San Francisco, San Francisco, CA, USA. AD - Institute for Computational Health Sciences, University of California, San Francisco, San Francisco, California, USA. FAU - Pollard, Katherine S AU - Pollard KS AD - Institute for Human Genetics, University of California, San Francisco, San Francisco, CA, USA. AD - Gladstone Institutes, San Francisco, CA, USA. AD - Chan-Zuckerberg Biohub, San Francisco, CA, USA. AD - Department of Epidemiology and Biostatistics and Institute of Computational Health Sciences, University of California, San Francisco, San Francisco, CA, USA. FAU - Shendure, Jay AU - Shendure J AUID- ORCID: 0000-0002-1516-1865 AD - Department of Genome Sciences, University of Washington, Seattle, WA, USA. shendure@uw.edu. AD - Howard Hughes Medical Institute, Seattle, WA, USA. shendure@uw.edu. AD - Brotman Baty Institute for Precision Medicine, University of Washington, Seattle, WA, USA. shendure@uw.edu. FAU - Kircher, Martin AU - Kircher M AUID- ORCID: 0000-0001-9278-5471 AD - Berlin Institute of Health (BIH), Berlin, Germany. martin.kircher@bihealth.de. AD - Charite-Universitatsmedizin Berlin, Berlin, Germany. martin.kircher@bihealth.de. FAU - Ahituv, Nadav AU - Ahituv N AUID- ORCID: 0000-0002-7434-8144 AD - Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, San Francisco, CA, USA. nadav.ahituv@ucsf.edu. AD - Institute for Human Genetics, University of California, San Francisco, San Francisco, CA, USA. nadav.ahituv@ucsf.edu. LA - eng GR - U01 MH116438/MH/NIMH NIH HHS/United States GR - T32 HL007093/HL/NHLBI NIH HHS/United States GR - UM1 HG009408/HG/NHGRI NIH HHS/United States GR - R21 HG010683/HG/NHGRI NIH HHS/United States GR - R01 GM142112/GM/NIGMS NIH HHS/United States GR - P30 AR070155/AR/NIAMS NIH HHS/United States GR - R21 HG010065/HG/NHGRI NIH HHS/United States GR - R01 MH116438/MH/NIMH NIH HHS/United States GR - U01 HG012192/HG/NHGRI NIH HHS/United States GR - HHMI/Howard Hughes Medical Institute/United States GR - F31 HG011007/HG/NHGRI NIH HHS/United States GR - R01 MH109907/MH/NIMH NIH HHS/United States GR - T32 GM067547/GM/NIGMS NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural PT - Research Support, Non-U.S. Gov't DEP - 20200708 PL - England TA - Nat Protoc JT - Nature protocols JID - 101284307 SB - IM EIN - Nat Protoc. 2021 Jul;16(7):3736. PMID: 33128032 MH - Base Sequence MH - Lentivirus/*genetics MH - Regulatory Sequences, Nucleic Acid/*genetics MH - Sequence Analysis, DNA/*methods MH - *Workflow PMC - PMC7550205 MID - NIHMS1632591 COIS- Competing interests The authors declare no competing interests. EDAT- 2020/07/10 06:00 MHDA- 2020/09/15 06:00 PMCR- 2021/02/01 CRDT- 2020/07/10 06:00 PHST- 2020/01/08 00:00 [received] PHST- 2020/04/17 00:00 [accepted] PHST- 2020/07/10 06:00 [pubmed] PHST- 2020/09/15 06:00 [medline] PHST- 2020/07/10 06:00 [entrez] PHST- 2021/02/01 00:00 [pmc-release] AID - 10.1038/s41596-020-0333-5 [pii] AID - 10.1038/s41596-020-0333-5 [doi] PST - ppublish SO - Nat Protoc. 2020 Aug;15(8):2387-2412. doi: 10.1038/s41596-020-0333-5. Epub 2020 Jul 8.