PRO-seq (Precision Run-On sequencing) to profile nascent RNA of H3.3 mutant (K9A; K27A; K79A) and control mESCs
PRO-seq (Precision Run-On sequencing) to profile nascent RNA of H3.3 mutant (K9A; K27A; K79A) and control mESCs
Mouse embryonic stem cells (129XC57BL/6J) were cultured in ESC medium containing Knockout-DMEM (Thermo Fisher Scientific) supplemented with 15% EmbryoMax fetal bovine serum (ES-FBS Merck-Millipore) and 20 ng/ml leukemia inhibitory factor (LIF, produced by Protein Expression and Purification Facility at EMBL Heidelberg), 1% non-essential amino acids (Thermo Fisher Scientific), 1% Glutamax (Thermo Fisher Scientific), 1% Pen/Strep (Thermo Fisher Scientific), 1% of 55 mM beta-Mercaptoethanol solution (Merck Millipore). Upon thawing, cells were cultured on a layer of mouse embryonic fibroblasts (MEFs) and afterwards passaged on 0.1% gelatine-coated culture dishes, without feeder cells. Cells were maintained at 37°C with 5% CO2 and routinely tested for mycoplasma. The medium was changed daily, and cells were passed every two days. Precision nuclear run-on transcription sequencing (PRO-seq) experiments were performed following the protocol previously described (PMID: 27442863). PRO-seq was performed in two batches, one with two replicates each of control and H3.3K27A mESCs and one with three replicates each of control and H3.3K9A mESCs. Cells were harvested by scraping and permeabilized on ice for 10 min in permeabilization buffer, followed by 1 wash with the permeabilization buffer and centrifugations at 1000x g at 4°C. Cells were DAPI-stained, counted and resuspended in storage buffer. Aliquots were snap-frozen and stored at -80°C. For the experiment, 10 million mESCs were used for each reaction and 5% (i.e., 500k) of permeabilized Drosophila S2 cells were  added prior to the in-vitro run-on reaction. Run-on reactions were carried out by adding 100 µl of 2× NRO reaction mixture (10 mM of Tris-HCl, pH 8.0; 5 mM MgCl2; 300 mM KCl; 1 mM dithiothreitol (DTT); 1% sarkosyl; 375 µM biotin-11-CTP/-UTP; 375  µM ATP/GTP; 2 µl RNase inhibitor) to 100 µl of the permeabilized cells, and incubated at 30 °C for 3 min. Addition of TRIzol LS (Thermo Fisher Scientific) terminated the reaction and RNA was extracted and precipitated in 75% ethanol. Nuclear RNA was fragmented by base hydrolysis in 0.2 N of NaOH on ice for 10 min and subsequently neutralized by adding the same volume of 1 M of Tris-HCl, pH 6.8. Fragmented biotinylated RNA was bound to the streptavidin magnetic beads (Thermo Fisher Scientific), washed, eluted using TRIzol and precipitated in ethanol. Following 3′–RNA and 5′–RNA adapter ligation, the precipitated RNA was reverse-transcribed and PCR-amplified. DNA libraries were purified using SPRI-select beads and quality was assessed on a Bioanalyzer using the Agilent High Sensitivity DNA Kit.