GSM8014790: Jurkat, IgG (PSIP1-matched control), CUT&RUN; Homo sapiens; OTHER
CUT&RUN was performed using CUTANA pA/G-MNase (Epicypher, 15-1016) according to manufacturer's instructions except for minor adaptations. Per sample, nuclei of 0.5M cells were extracted (20mM HEPES-KOH pH 7.9, 10mM KCl, 0.1% Triton X-100, 20% glycerol 1mM MnCl2, 0.5mM spermidine and protease inhibitors), incubated for 10 min at 4°C on a rotator with activated concanavalin A beads, resuspended in antibody buffer (150mM NaCl, 20mM HEPES-KOH pH 7.5, 2mM EDTA, 0.5mM spermidine and protease inhibitors) and incubated overnight at 4°C with rotation with 1:100 dilution of antibody recognizing PSIP1 (Bethyl, A300-848A), H3K27me3 (Cell Signaling Technology, 97335), H3K36me3 (Abcam, ab9050) or a rabbit anti-IgG (Cell Signaling Technology, 3900S). Targeted chromatin digestion and release was performed with 2.5 µl CUTANA pA/G-MNase and 100 mM CaCl2. The reaction was terminated with STOP buffer (340 mM NaCl, 4mM EGTA, 20mM EDTA, RNAase (0.05 mg/ml) and glycogen (0.05 µg/ml). For the mice samples, 0.01 ng/ml spike-in E.Coli DNA (Epicypher, 18-1401) was added. Retrieved genomic DNA was purified with the CUTANA DNA purification kit V2 (Epicypher, 14-0050). Smples were library prepped using the NEBNext Ultra II DNA Library Prep Kit according to the manufacturing's instructions, but NEBNext Endprep was conducted at 50°C for 1h. Sequencing was done on the AVITI, 2x75 (Element Biosciences).