title: GSM9458906: Mus musculus trophoblast H3K4me1, rep 1; Mus musculus; OTHER
library_construction_protocol: CUT&Tag was performed using either 50,000 cells or 5 mg of tissue per antibody. Cells were harvested fresh using TrypLE Express and centrifuged at 600 × g for 5 min at room temperature (RT). Placentas were homogenised in 1 ml wash buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 1X protease inhibitor cocktail (PIC), 0.1% BSA) with a Dounce homogeniser using a tight pestle 30 times. Dounced tissue was transferred to a 1.5 ml tube and centrifuged at 600 × g for 5 min at RT. Pellet was resuspended in 500 µl ice-cold nuclei extraction buffer (20 mM HEPES pH 7.5, 10 mM KCl, 0.5 mM spermidine, 1% Triton X-100, 20% glycerol, 1X PIC) and incubated on ice for 10 minutes. Nuclei were pelleted by centrifugation at 1,300 × g for 5 min at 4 ˚C. Pelleted cells or nuclei were washed twice with 1 ml wash buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 1X PIC) by gentle pipetting. BioMagPlus Concanavalin A-coated magnetic beads (Generon) were activated by washing and resuspension in binding buffer (20 mM HEPES pH 7.5, 10 mM KCl, 1 mM CaCl₂, 1 mM MnCl₂). Activated beads (10 µL per sample) were added to the cells and incubated for 10 min at RT. Bead-bound cells were isolated using a magnetic stand, the supernatant was removed, and cells were resuspended in 100 µl antibody buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 1X PIC, 0.05% digitonin, 2 mM EDTA, 0.1% BSA). Primary antibodies (H3K4me1 [Abcam ab8895], H3K4me3 [Active Motif 39060], H3K27ac [Abcam ab4729], or IgG [Santa Cruz sc-2027]) were added at a 1:100 dilution, and samples were incubated overnight at 4 °C on a nutator. Cells were incubated with secondary antibody (guinea pig anti-rabbit IgG [ABIN101961]) diluted 1:100 in 100 µl Dig-Wash buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 1X PIC, 0.05% digitonin) for 1 h at RT on a nutator. Cells were washed three times with 800 µl Dig-Wash buffer before incubation with Protein A-Tn5 transposase fusion protein (pA-Tn5), pre-loaded with Illumina Nextera adapters (kind gift from the Henikoff laboratory via the Madapura laboratory). pA-Tn5 was diluted 1:250 in Dig-300 buffer (20 mM HEPES pH 7.5, 300 mM NaCl, 0.5 mM spermidine, 1X PIC, 0.05% digitonin), and 100 µl was added per sample followed by incubation for 1 h at RT on a nutator. Unbound pA-Tn5 was removed by three washes with 800 µL Dig-300 buffer. Cells were then resuspended in 300 µl tagmentation buffer (10 mM MgCl₂ in Dig-300 buffer) and incubated at 37 °C for 1 h on a nutator. Tagmentation was stopped by the addition of 10 µl 0.5 M EDTA, 3 µl 10% SDS, and 2.5 µl Proteinase K (20 mg/ml), followed by overnight incubation at 37 °C. DNA was extracted using phenol:chloroform:isoamyl alcohol (25:24:1, v/v; Sigma-Aldrich) and Phase Lock Gel Heavy tubes (5 PRIME), followed by chloroform extraction and ethanol precipitation. DNA pellets were washed with 100% ethanol, air-dried, and resuspended in 30 µl TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA) containing RNase A (1:400). Libraries were amplified using 8 µl DNA per sample with NEBNext HiFi 2X PCR Master Mix, universal i5 primer, and uniquely indexed i7 primers (0.5 µM each). PCR conditions were 72 °C for 5 min, 98 °C for 30 s, followed by 12 cycles of 98 °C for 10 s and 63 °C for 10 s, with a final extension at 72 °C for 1 min. Libraries were purified using AMPure XP beads (Beckman Coulter) according to the manufacturer's instructions.