RNA Polymerase II hypertranscription in cancer FFPE samples.

Hypertranscription is common in human cancers and predicts poor prognosis. However detection of hypertranscription is indirect, relying on accurately quantifying mRNA levels and estimating cell numbers. Previously, we introduced FFPE-CUTAC, a genome-wide method for mapping RNA Polymerase II (RNAPII) in formalin-fixed paraffin-embedded (FFPE) sections. Here we use FFPE-CUTAC to demonstrate genome-wide hypertranscription both in transgene-driven mouse gliomas and in assorted human tumors at active regulatory elements and replication-coupled histone genes with reduced mitochondrial DNA abundance. FFPE-CUTAC identified RNAPII-bound regulatory elements shared among diverse cancers and readily categorized human tumors despite using very small samples and low sequencing depths. Remarkably, RNAPII FFPE-CUTAC identified de novo and precisely mapped HER2 amplifications punctuated by likely selective sweeps including genes encoding direct positive regulators of RNAPII itself. Our results demonstrate that FFPE-CUTAC measurements of hypertranscription and classifications of tumors using small sections provides an affordable and sensitive genome-wide strategy for personalized medicine.

Scalable single-cell profiling of chromatin modifications with sciCUT&Tag.

Cleavage under targets and tagmentation (CUT&Tag) is an antibody-directed in situ chromatin profiling strategy that is rapidly replacing immune precipitation-based methods, such as chromatin immunoprecipitation-sequencing. The efficiency of the method enables chromatin profiling in single cells but is limited by the numbers of cells that can be profiled. Here, we describe a combinatorial barcoding strategy for CUT&Tag that harnesses a nanowell dispenser for simple, high-resolution, high-throughput, single-cell chromatin profiling. In this single-cell combinatorial indexing CUT&Tag (sciCUT&Tag) protocol, lightly cross-linked nuclei are bound to magnetic beads and incubated with primary and secondary antibodies in bulk and then arrayed in a 96-well plate for a first round of cellular indexing by antibody-directed Tn5 tagmentation. The sample is then repooled, mixed and arrayed across 5,184 nanowells at a density of 12-24 nuclei per well for a second round of cellular indexing during PCR amplification of the sequencing-ready library. This protocol can be completed in 1.5 days by a research technician, and we illustrate the optimized protocol by profiling histone modifications associated with developmental gene repression (H3K27me3) as well as transcriptional activation (H3K4me1-2-3) in human peripheral blood mononuclear cells and use single-nucleotide polymorphisms to facilitate collision removal. We have also used sciCUT&Tag for simultaneous profiling of multiple chromatin epitopes in single cells. The reduced cost, improved resolution and scalability of sciCUT&Tag make it an attractive platform to profile chromatin features in single cells.