not depend on prior knowledge of what transcripts to study (as microarrays do). RNA sequencing RNA sequencing has taken over microarray and SAGE technology in recent years, as noted in 2016, and has become the most efficient way to study transcription and gene expression. This is typically done by next-generation sequencing. A subset of sequenced RNAs are small RNAs, a class of non-coding RNA molecules that are key regulators of transcriptional and post-transcriptional gene silencing, or RNA silencing. Next-generation sequencing is the gold standard tool for non-coding RNA discovery, profiling and expression analysis. Massively Parallel Reporter Assays (MPRAs) Massively parallel reporter assays is a technology to test the cis-regulatory activity of DNA sequences. MPRAs use a plasmid with a synthetic cis-regulatory element upstream of a promoter driving a synthetic gene such as Green Fluorescent Protein. A library of cis-regulatory elements is usually tested using MPRAs, a library can contain from hundreds to thousands of cis-regulatory elements. The cis-regulatory activity of the elements is assayed by using the downstream reporter activity. The activity of all the library members is assayed in parallel using barcodes for each cis-regulatory element. One limitation of MPRAs is that the activity is assayed on a plasmid and may not capture all aspects of gene regulation observed in the genome. STARR-seq STARR-seq is a technique similar to MPRAs to assay enhancer activity of randomly sheared genomic fragments. In the original publication, randomly sheared fragments of the Drosophila genome were placed downstream of a minimal promoter. Candidate enhancers amongst the randomly sheared fragments will transcribe themselves using the minimal promoter. By using sequencing as a readout and controlling for input amounts of each sequence the strength of putative enhancers are assayed by this method. Perturb-seq Perturb-seq couples CRISPR mediated gene knockdowns with single-cell gene expression. Linear models are used to calculate the effect of the knockdown of a single gene on the expression of multiple genes. At the protein level Yeast two-hybrid system A yeast two-hybrid screening (Y2H) tests a "bait" protein against many potential interacting proteins ("prey") to identify physical protein–protein interactions. This system is based on a transcription factor, originally GAL4, whose separate DNA-binding and transcription activation domains are both required in order for the protein to cause transcription of a reporter gene. In a Y2H screen, the "bait" protein is fused to the binding domain of GAL4, and a library of potential "prey" (interacting) proteins