ORE-seq: genome-wide absolute occupancy measurement by restriction enzyme accessibilities

Digestion with restriction enzymes is a classical approach for probing DNA accessibility in chromatin. It allows to monitor both the cut and the uncut fraction and thereby the determination of accessibility or occupancy (= 1- accessibility) in absolute terms as the percentage of cut or uncut out of the total molecules. The here presented protocol takes this classical approach to the genome-wide level. After exhaustive restriction enzyme digestion of chromatin, DNA is purified, sheared and converted into libraries for high throughput sequencing. Bioinformatic analysis counts DNA fragments cut by the restriction enzyme as well as DNA ends generated by restriction enzyme digest and derives thereof the fraction of accessible DNA. This straight forward principle is technically challenged as preparation and sequencing of the libraries leads to biased scoring of DNA fragments with ends generated by restriction enzymes versus by shearing. Our protocol includes two orthogonal approaches to correct for this bias, the “corrected cut-uncut” and the “cut-all cut” method, so that accurate measurements of absolute accessibility/occupancy at restriction sites throughout a genome are possible. The protocol is presented for the example of S. cerevisiae chromatin but may be adapted for any other species.限制性内切酶（restriction enzymes）酶切是检测染色质中DNA可及性的经典实验方法。该方法可同时监测被酶切与未被酶切的DNA组分，进而以总分子中被酶切或未被酶切组分的占比作为绝对量化依据，计算得到DNA可及性或占用率（occupancy，等同于1减去可及性）。本研究提出的实验方案将这一经典方法拓展至全基因组层面。在对染色质进行彻底的限制性内切酶酶切后，对DNA进行纯化、断裂处理，并构建用于高通量测序的测序文库。通过生物信息学分析，统计经限制性内切酶切割产生的DNA片段以及限制性酶切生成的DNA末端，进而推导得到可及DNA的占比。这一原理看似简单直接，但在技术层面存在局限：文库制备与测序过程中，相较于机械断裂产生的DNA末端，经限制性酶切生成的DNA末端对应的片段会出现评分偏倚。本方案采用两种正交校正方法来抵消该偏倚，即"corrected cut-uncut"法与"cut-all cut"法，从而实现全基因组范围内限制性酶切位点处绝对可及性/占用率的精准定量。本方案以酿酒酵母（Saccharomyces cerevisiae）染色质为例进行展示，但同样可适配其他任意物种。