24Tracking active transcription with the nuclear run-on (NRO) assays has been instrumental in 25 uncovering mechanisms of gene regulation. The coupling of NROs with high-throughput 26 sequencing has facilitated the discovery of previously unannotated or undetectable RNA classes 27 genome-wide. Precision run-on sequencing (PRO-seq) is a run-on variant that maps polymerase 28 active sites with nucleotide or near-nucleotide resolution. One main drawback to this and many 29 other nascent RNA detection methods is the somewhat intimidating multi-day workflow 30 associated with creating the libraries suitable for high-throughput sequencing. Here, we present 31an improved PRO-seq protocol where many of the enzymatic steps are carried out while the 32 biotinylated NRO RNA remains bound to streptavidin-coated magnetic beads. These 33 adaptations reduce time, sample loss and RNA degradation, and we demonstrate that the 34 resulting libraries are of the same quality as libraries generated using the original published 35protocol. The assay is also more sensitive which permits reproducible, high-quality libraries from 36 10 4 -10 5 cells instead of 10 6 -10 7 . Altogether, the improved protocol is more tractable allows for 37 nascent RNA profiling from small samples, such as rare samples or FACS sorted cell 38populations. 39 40
Chronic lymphocytic leukemia (CLL) is the most common leukemia in Western countries. CLL remains incurable despite improvements in clinical outcomes from the identification of prognostic markers and the introduction of targeted therapies. Recent studies have identified differences in the epigenetic and the regulatory landscape of CLL that may provide molecular targets for future therapies. Optical genome mapping (OGM) is a new method that may improve clinical testing and CLL patient care because it can provide greater sensitivity and resolution of structural variation (SV) that is currently detected by chromosome banding analysis (CBA). The practical issues around diagnosis, molecular cytogenetic prognostic markers, pathobiology, and targeted therapies are discussed with brief reference to OGM.
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