PRO-seq (Precision Run-On sequencing) to profile nascent RNA of H3.3 mutant (K9A; K27A; K79A) and control mESCs

Mouse embryonic stem cells (i.e., mESCs; line ESC 129-B13) were genetically modified using CRISPR-Cas9 to mutate the H3f3b locus, in order to carry homozygous lysine-to-alanine substitution of residues K9 or K27. Two control mESC lines carrying knock-out of the H3f3a gene were used as background for the editing. To profile nascent RNA levels in H3.3 mutant and control mESCs, two rounds of PRO-seq experiment were performed. In the first round, three replicates each of H3.3K9A and control (i.e., Ctrl_9) mESCs were used. In the second round, two replicates each of H3.3K27A and control (i.e., Ctrl_27) mESCs were used. After permeabilization of the mESCs (and addition of 5% Drosophila S2 cells spike-in), a 2 biotin run-on reaction using biotin-11-UTP and biotin-11-CTP was carried out to label nascent transcripts. Biotinylated nascent RNA was then enriched as part of the library preparation process. Sequencing was performed on a NextSeq500 platform in single-end mode (75bp reads). The experiment allows for an assesment of the changes in the activity of cis-regulatory elements, upon removal of H3.3 K9 or K27 residues.