An essential group of non-coding genome elements are enhancers which can affect gene expression in an orientation and position independent manner. This means that enhancers can regulate genes even when they are located far away or in a reverse orientation compared to the gene itself. A few methods, such as chromatin immunoprecipitation abided by sequencing (known as ChIP-seq) and chromatin accessibility assays can foretell the position of enhancers. Despite these means to predict enhancer locations, is it is very challenging to predict the activity of these sequences. Based on previous research of the host lab (Barakat et al., 2017), a novel experimental approach was developed which enables the identification of enhancer functionality on a genome-wide scale in a quantitative manner. Using this combination of ChIP and a massively-parallel-reporter assay system called STARR-seq, this ChIP-STARR-seq discovered that only a small fraction of genomic regions marked by histone modifications or bound by transcription factors do show measurable enhancer activity in human embryonic stem cells (hESC). During differentiation, enhancer activity changes rapidly and only small components of “super-enhancers” are responsible for enhancer activity. In order to investigate the role of enhancers in cells for neurodevelopmental disorders a genome-wide functional assay called ChIP-STARR-seq (Figure 5) is proposed. According to the publication in “Current protocols in molecular biology” journal the ChIP-seq (Figure 3) methodology involves chromatin immunoprecipitation abided by high-efficiency sequencing (Raha, Hong, & Snyder, 2010).
Thereafter chromatin is isolated from nuclei and subjected to sonication which is applied during an ultrasonic bath or ultrasonic probe called sonicator. The sonication process is based on the conversion of an electrical signal into a physical vibration that is directed towards a substance. This is needed for breaking apart compounds or cells for further analysis, and shearing of the DNA into smaller pieces ranging from 200 to 600 bp. Next, an antibody is used to immunoprecipitate specific DNA-transcription factor complexes or histone modifications from the isolated chromatin. After the immunoprecipitation, co-immunoprecipitated DNA is purified and sequencing adapters are ligated. For a normal ChIP-seq, this material would now be sequenced to determine the location of the pulled-down sequences in the human genome. In the ChIP-STARR-seq approach this also occurs, but in addition, the adapter-ligated material is also used to clone reporter plasmid libraries. After the library preparation is done transfection of target cells is taking place. Transfection is done by introducing a foreign DNA into the nucleus of eukaryotic cells. There are some important factors to take into account which may affect transfection (BIO-RAD, 2017). Several of the essential points is to use appropriate medium, keep cultures free of contamination, maintain cells in log phase growth and use high-quality plasmid DNA that is free of proteins and RNA.