title: GSM5825565: XEN_XX_H3K27me3_r3; Mus musculus; OTHER
library_construction_protocol: For each CUT&Tag reaction 100.000 cells were collected and washed once with Wash buffer (20 mM HEPES-KOH, pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, 10 mM Sodium butyrate, 1 mM PMSF). 10ul Concanavalin A (Bangs Laboratories) beads were equilibrated with 100ul Binding buffer (20 mM HEPES-KOH, pH 7.5, 10 mM KCl, 1 mM CaCl2, 1mM MnCl2) and afterwards concentrated in 10ul binding buffer. The cells were bound to the Concanavalin A beads  by incubating for 10 minutes at room temperature with rotation. Following this, the beads were separated on a magnet and resuspended in 100ul chilled Antibody buffer (Wash buffer with 0.05% Digitonin and 2mM EDTA) with subsequently 1ul of primary antibody and incubated on a rotator for 3h at 4°C. Primary antibodies were used 1:100 (H3K27ac, H3K27me3) or 1:200 (GATA2/3/4/6).  After magnetic separation the beads were resuspended in 100ul chilled Dig-wash buffer (Wash buffer with 0.05% Digitonin) containing 1ul of matching secondary antibodies and were incubated for 1h at 4°C with rotation. The beads were washed three times with ice cold Dig-wash buffer and resuspended in chilled Dig-300 buffer (20 mM HEPES-KOH, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 0.01% Digitonin, 10 mM Sodium butyrate, 1 mM PMSF) with 1:250 diluted 3xFLAG-pA-Tn5 preloaded with Mosaic-end adapters. After incubation for 1h at 4°C with rotation, the beads were washed four times with chilled Dig-300 buffer and resuspended in 50ul Tagmentation buffer (Dig-300 buffer 10 Mm MgCl2). Tagmentation was performed for 1h at 37°C and subsequently stopped by adding 2.25 µL 0.5M EDTA, 2.75 µL 10% SDS and 0.5 µL 20 mg/mL Proteinase K and vortexing for 5 seconds. DNA fragments were solubilized overnight at 55°C followed by 30 minutes at 70°C to inactivate residual Proteinase K. DNA fragments were purified with the ChIP DNA Clean & Concentrator kit (Zymo Research) and eluted with 25 ul elution buffer according to the manufacturer's guidelines. NGS libraries were generated by amplifying the CUT&Tag DNA fragments with i5 and i7 Nextera barcoded HPLC grade primers using NEBNext® HiFi 2x PCR Master Mix (New England BioLabs) on a thermocycler with the following program: 72 °C for 5 min, 98 °C for 30 sec, 98 °C for 10 sec, 63 °C for 10 sec (14-15 Cycles for step 3-4)  and 72°C for 1 min. Post PCR cleanup was performed with Ampure XP beads (Beckman Coulter). For this 0.95x volume of Ampure XP beads were mixed with the NGS libraries and incubated at room temperature for 10 minutes. After magnetic separation, the beads were washed three times on the magnet with 80% ethanol and the libraries eluted with Tris-HCl, pH 8.0. The quality of the purified NGS libraries was assessed with the BioAnalyzer High Sensitivity DNA system (Agilent Technologies). Sequencing libraries were pooled in equimolar ratios cleaned for another round with  1.2x volume of Ampure XP beads as before and eluted in 20ul Tris-HCl, pH 8.0. The sequencing library pool quality was assessed with the BioAnalyzer High Sensitivity DNA system (Agilent Technologies) and subjected to Illumina PE75 next generation sequencing on the NextSeq500 platform to approximately 5 million fragments per library. CUT&Tag