lentiMPRA and MPRAflow for high-throughput functional characterization of gene regulatory elements

M Grace Gordon, Fumitaka Inoue, Beth Martin, Max Schubach, Vikram Agarwal, Sean Whalen, Shiyun Feng, Jingjing Zhao, Tal Ashuach, Ryan Ziffra, Anat Kreimer, Ilias Georgakopoulos-Soares, Nir Yosef, Chun Jimmie Ye, Katherine S Pollard, Jay Shendure, Martin Kircher, Nadav Ahituv

نتاج البحث: نشر في مجلةمقالةمراجعة النظراء

ملخص

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.
اللغة الأصليةالإنجليزيّة
الصفحات (من إلى)2387-2412
عدد الصفحات28
دوريةNATURE PROTOCOLS
مستوى الصوت15
رقم الإصدار8
تاريخ مبكر على الإنترنت8 يوليو 2020
المعرِّفات الرقمية للأشياء
حالة النشرنُشِر - أغسطس 2020
منشور خارجيًانعم

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