lentiMPRA and MPRAflow for high-throughput functional characterization of gene regulatory elements.,Nature Protocols

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lentiMPRA and MPRAflow for high-throughput functional characterization of gene regulatory elements.
Nature Protocols ( IF 13.1 ) Pub Date : 2020-07-08 , DOI: 10.1038/s41596-020-0333-5
M Grace Gordon _{1,

2,

3} , Fumitaka Inoue _{1,

2} , Beth Martin ₄ , Max Schubach _{5,

6} , Vikram Agarwal _{4,

7} , Sean Whalen ₈ , Shiyun Feng _{1,

2} , Jingjing Zhao _{1,

2} , Tal Ashuach ₉ , Ryan Ziffra _{1,

2} , Anat Kreimer _{1,

2,

9} , Ilias Georgakopoulos-Soares _{1,

2} , Nir Yosef _{9,

10} , Chun Jimmie Ye _{1,

2,

10,

11,

12} , Katherine S Pollard _{2,

8,

10,

13} , Jay Shendure _{4,

14,

15} , Martin Kircher _{5,

6} , Nadav Ahituv _{1,

2}

Affiliation

Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, San Francisco, CA, USA.
Institute for Human Genetics, University of California, San Francisco, San Francisco, CA, USA.
Biological and Medical Informatics Graduate Program, University of California, San Francisco, San Francisco, CA, USA.
Department of Genome Sciences, University of Washington, Seattle, WA, USA.
Berlin Institute of Health (BIH), Berlin, Germany.
Charité-Universitätsmedizin Berlin, Berlin, Germany.
Calico Life Sciences LLC, South San Francisco, CA, USA.
Gladstone Institutes, San Francisco, CA, USA.
Department of Electrical Engineering and Computer Sciences and Center for Computational Biology, University of California, Berkeley, Berkeley, CA, USA.
Chan-Zuckerberg Biohub, San Francisco, CA, USA.
Division of Rheumatology, Department of Medicine, University of California, San Francisco, San Francisco, CA, USA.
Institute for Computational Health Sciences, University of California, San Francisco, San Francisco, California, USA.
Department of Epidemiology and Biostatistics and Institute of Computational Health Sciences, University of California, San Francisco, San Francisco, CA, USA.
Howard Hughes Medical Institute, Seattle, WA, USA.
Brotman Baty Institute for Precision Medicine, University of Washington, Seattle, WA, USA.

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.

中文翻译：

lentiMPRA 和 MPRAflow 用于基因调控元件的高通量功能表征。

大规模并行报告基因检测 (MPRA) 可以同时定量测量数千个候选调控序列 (CRS) 的功能。在这种方法中，CRS 被克隆到最小启动子和报告基因的上游，以及独特的条形码，并引入细胞中。如果 CRS 是功能性调控元件，它将导致条形码序列的转录，该转录通过 RNA 测序进行测量，并通过条形码的 DNA 测序对细胞整合进行标准化。该技术已用于测试数千个序列及其变体的调节活性，破译调节密码及其进化，以及开发基因开关。基于慢病毒的 MPRA (lentiMPRA) 产生“基因组内”读数，并能够在难以转染的细胞中使用该技术。在这里，我们提供了 lentiMPRA 的详细协议，以及用户友好的基于 Nextflow 的计算管道 - MPRAflow - 用于量化不同 MPRA 设计的 CRS 活动。 lentiMPRA 协议大约需要 2 个月，其中包括测序周转时间和使用 MPRAflow 进行数据处理。

更新日期：2020-07-08

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