Enhancer RNAs (eRNA) are unstable non-coding RNAs, transcribed bidirectionally from active regulatory sequences, whose expression levels correlate with enhancer activity. We use capped-nascent-RNA sequencing to efficiently capture bidirectional transcription initiation across several human lymphoblastoid cell lines (Yoruba population) and detect ~75,000 eRNA transcription sites with high sensitivity and specificity. The use of nascent-RNA sequencing sidesteps the confounding effect of eRNA instability. We identify quantitative trait loci (QTLs) associated with the level and directionality of eRNA expression. High-resolution analyses of these two types of QTLs reveal distinct positions of enrichment at the central transcription factor (TF) binding regions and at the flanking eRNA initiation regions, both of which are associated with mRNA expression QTLs. These two regions—the central TF-binding footprint and the eRNA initiation cores—define a bipartite architecture of enhancers, inform enhancer function, and can be used as an indicator of the significance of non-coding regulatory variants.

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

Nuclear Run-On sequencing is a powerful method to measure transcription with high resolution, sensitivity, and directional information, which provides alternative perspective from existing methods such as chromatin immunoprecipitation or mRNA sequencing. Current form of Nuclear Run-On assays such as Precision Run-On sequencing (PRO-seq) involves multiple RNA chemistry steps including RNA end repairs and ligations. These have limited the widespread use of PRO-seq by requiring robust RNA handling skills and multiple days of effort. To solve this, we developed an ultrashort PRO-seq (uPRO) method that requires minimal steps. In uPRO, the requirement of only two reactions -RNA adaptor ligation and template switch reverse transcriptionreduced the procedure into less than a single day. Using uPRO, we generated genome-wide transcription profiles of human haploid cell lines (HAP1) and peripheral blood samples combined with Chromatin Run-On sequencing (pChRO). Blood cell handling procedure is dramatically reduced using pChRO directly on crude chromatin preparations, and enables utilizing archived specimens. As a result, we identified individual differences in the transcriptional profiles of human whole blood from a small volume (~1 ml). We also generated blood cell type specific transcription data, and deconvoluted the nucleated blood cell compositions by modeling to the reference datasets. Overall, uPRO and pChRO provided a powerful platform to identify differentially expressed genes between individuals with minimal sample requirements. Fig 1. Schematics of the uPRO procedure A. Comparison between conventional PRO-seq and uPRO procedures. Adapted from Mahat et al 12