Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, USA.
The Baker Institute of Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, New York, USA.
Nat Protoc. 2016 Aug;11(8):1455-76. doi: 10.1038/nprot.2016.086. Epub 2016 Jul 21.
We provide a protocol for precision nuclear run-on sequencing (PRO-seq) and its variant, PRO-cap, which map the location of active RNA polymerases (PRO-seq) or transcription start sites (TSSs) (PRO-cap) genome-wide at high resolution. The density of RNA polymerases at a particular genomic locus directly reflects the level of nascent transcription at that region. Nuclei are isolated from cells and, under nuclear run-on conditions, transcriptionally engaged RNA polymerases incorporate one or, at most, a few biotin-labeled nucleotide triphosphates (biotin-NTPs) into the 3' end of nascent RNA. The biotin-labeled nascent RNA is used to prepare sequencing libraries, which are sequenced from the 3' end to provide high-resolution positional information for the RNA polymerases. PRO-seq provides much higher sensitivity than ChIP-seq, and it generates a much larger fraction of usable sequence reads than ChIP-seq or NET-seq (native elongating transcript sequencing). Similarly to NET-seq, PRO-seq maps the RNA polymerase at up to base-pair resolution with strand specificity, but unlike NET-seq it does not require immunoprecipitation. With the protocol provided here, PRO-seq (or PRO-cap) libraries for high-throughput sequencing can be generated in 4-5 working days. The method has been applied to human, mouse, Drosophila melanogaster and Caenorhabditis elegans cells and, with slight modifications, to yeast.
我们提供了一种精确核 RNA 转录延伸测序（PRO-seq）及其变体 PRO-cap 的方案，该方案可在高分辨率下全局绘制活跃 RNA 聚合酶（PRO-seq）或转录起始位点（TSS）（PRO-cap）的位置。特定基因组位置的 RNA 聚合酶密度直接反映了该区域新生转录的水平。从细胞中分离细胞核，并在核转录延伸条件下，参与转录的 RNA 聚合酶将一个或最多几个生物素标记的核苷酸三磷酸（biotin-NTP）掺入新生 RNA 的 3' 端。生物素标记的新生 RNA 用于制备测序文库，这些文库从 3' 端测序，为 RNA 聚合酶提供高分辨率的位置信息。PRO-seq 的灵敏度比 ChIP-seq 高得多，并且它产生的可用序列读取片段比 ChIP-seq 或 NET-seq（天然延伸转录测序）多得多。与 NET-seq 类似，PRO-seq 以碱基对分辨率并具有链特异性地绘制 RNA 聚合酶的图谱，但与 NET-seq 不同，它不需要免疫沉淀。使用这里提供的方案，可以在 4-5 个工作日内生成用于高通量测序的 PRO-seq（或 PRO-cap）文库。该方法已应用于人类、小鼠、黑腹果蝇和秀丽隐杆线虫细胞，并经过轻微修改后也应用于酵母。
