Gordon M Grace, Inoue Fumitaka, Martin Beth, Schubach Max, Agarwal Vikram, Whalen Sean, Feng Shiyun, Zhao Jingjing, Ashuach Tal, Ziffra Ryan, Kreimer Anat, Georgakopoulos-Soares Ilias, Yosef Nir, Ye Chun Jimmie, Pollard Katherine S, Shendure Jay, Kircher Martin, Ahituv Nadav
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.
Nat Protoc. 2020 Aug;15(8):2387-2412. doi: 10.1038/s41596-020-0333-5. Epub 2020 Jul 8.
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.
大规模平行报告基因检测（MPRAs）能够以定量方式同时测量数千个候选调控序列（CRSs）的功能。在该方法中，CRSs被克隆到最小启动子和报告基因的上游，并与一个独特的条形码一起导入细胞。如果CRS是一个功能性调控元件，它将导致条形码序列的转录，通过RNA测序对其进行测量，并通过条形码的DNA测序对细胞整合进行标准化。这项技术已被用于测试数千个序列及其变体的调控活性，以破译调控密码及其进化，并开发基因开关。基于慢病毒的MPRA（lentiMPRA）产生“基因组内”读数，并使该技术能够在难以转染的细胞中使用。在这里，我们提供了一份详细的lentiMPRA方案，以及一个基于Nextflow的用户友好型计算管道——MPRAflow，用于从不同的MPRA设计中量化CRS活性。lentiMPRA方案大约需要2个月时间，这包括测序周转时间和使用MPRAflow进行数据处理。
