GSM8721566: D16_ATRA_H3K27ac; Mus musculus; OTHER
Cells differentiated during 16 days of treatment were collected as dry pellets and used for Cut&Tag experiments were carried out as previously described (Kaya-Okur et al; Li et al), with some modifications. Briefly, 250.000 cells were captured with BioMagPlus Concanavalin A (Polysciences, 86057-3) and incubated with the appropriate primary antibody overnight at 4°C (anti-Histone H3 (tri methyl K4) antibody (Abcam, ab8580; 1:50); anti-Histone H3(acetyl K27) antibody (Abcam, ab4729; 1:50); anti-Histone H3(tri methyl K27) antibody (Abcam, ab192985; 1:50); Anti- RNA Pol II monoclonal antibody: diagenode (C15100055) (1:20)). For the experiment in which the antibody RNA Pol II was used, the mixture cells-beads were fixed with 1% PFA in 1xPBS for 15 minutes before the addition of the primary antibody. A secondary antibody was incubated at room temperature for 1 hour (Donkey anti-rabbit IgG antibody: Sigma-Aldrich, SAB3700932; 1:100). Fusion protein ProteinA-Tn5 was prepared in-house (plasmid Addgene #124601) by following a previously described protocol (Li et al). Purified PA-Tn5 was loaded with the following adapters compatible with Illumina sequencing (hybridized with the complementary Mosaic sequence [PHO]CTGTCTCTTATACACATCT): MOS-universal: TACACTCTTTCCCTACACGACGCTCTTCCGATCTAGATGTGTATAAGAGACAG MOS-index: GTTCAGACGTGT.GCTCTTCCGATCTAGATGTGTATAAGAGACAG Loaded PA-Tn5 complex (0.16 μM) was incubated for 1 hour at RT, followed by tagmentation step for 2 hours at 37°C. DNA was extracted by phenol/chloroform (Sigma-Aldrich 77617) followed by ethanol precipitation. Finally, the tagmented DNA was amplified using Illumina primers (NEBNext® Multiplex Oligos for Illumina, NEB #7335S) with the following cycling conditions: 98°C for 30s, 20 cycles of 98°C for 10s and 65°C for 75 s; then final extension at 65°C for 5 min and hold at 12°C. DNA libraries were cleaned using 1x volume of SPRI-select beads (Beckman Coulter, B23318) and Illumina sequenced (150-nt pair-end sequencing; Illumina NovaSeq 6000).