lentiMPRA and MPRAflow for high-throughput functional characterization of gene regulatory elements.
M Grace GordonFumitaka InoueBeth MartinMax SchubachVikram AgarwalSean WhalenShiyun FengJingjing ZhaoTal AshuachRyan ZiffraAnat KreimerIlias Georgakopoulos-SoaresNir YosefChun Jimmie YeKatherine S PollardJay ShendureMartin KircherNadav AhituvPublished in: Nature protocols (2020)
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.
Keyphrases
- transcription factor
- high throughput
- single cell
- induced apoptosis
- cell cycle arrest
- genome wide
- copy number
- randomized controlled trial
- crispr cas
- dna methylation
- gene expression
- signaling pathway
- electronic health record
- single molecule
- machine learning
- endoplasmic reticulum stress
- cell proliferation
- cell free
- genome wide identification
- big data
- mass spectrometry
- pi k akt
- artificial intelligence