GSM9248518: NPPO-2, H3K36me2, Cut&Tag; Mus musculus; OTHER
Organoids were dissociated into single cells using TrypLE, counted using a TC20 automated cell counter, and 200,000 cells/sample used for profiling. For experiments requiring normalization of input DNA, 10,000 Drosophila Schneider 2 (S2) cells (ThermoFisher Scientific R69007) were combined with 200,000 experimental cells at a ratio of 1:20. After centrifugation at 1000 rpm at RT, cell pellets were washed once with 1 ml wash buffer (20 mM HEPES (pH 7.5), 150 mM NaCl, 0.5 mM spermidine (Millipore-Sigma S2501), and cOmplete™, EDTA-free protease inhibitor cocktail (Millipore-Sigma 11873580001) in nuclease-free water). Cell pellets were resuspended with 10 µl ConA beads in 0.4 ml wash buffer and incubated for 10 minutes at RT. The tubes were then placed on a magnetic stand (New England Biolabs S1515S) to clear and remove the liquid, with pellets resuspended with 50 µl ice-cold Dig-wash buffer (wash buffer with 0.05% digitonin) and placed on ice for 10 min to lyse the cells. The samples were then transferred to an 8-strip tube (EpiCypher 10-0009). Each sample was incubated with 0.5 µg primary or IgG control antibody (Supplementary Table 6) and placed on a nutator at 4°C overnight. The next day, samples were incubated with 1 µl secondary antibody in 50 µl ice-cold Dig-wash buffer on a nutator at RT for 1 hr. After washing with Dig-Wash buffer three times, beads were resuspended with 50 µl Dig-300 buffer (20 mM HEPES (pH 7.5), 300 mM NaCl, 0.5 mM Spermidine, 0.01% Digitonin, cOmplete™, EDTA-free Protease Inhibitor Cocktail in nuclease-free water) containing 2.5 µl pA-Tn5 adapter complex (EpiCypher 15-1017) and placed on a nutator at RT for 1 hour. After washing three times with Dig-300 buffer, the beads were resuspended in 150 µl Tagmentation buffer (Dig-300 buffer with 10 mM MgCl2) and incubated at 37°C for 1 hr. To stop tagmentation and solubilize DNA fragments, 5 µL 0.5 M EDTA (ThermoFisher Scientific AM9260G), 1.5 µl 10% SDS (Millipore-Sigma 71736), 1.25 µl 20 mg/ml Proteinase K (ThermoFisher Scientific EO0491) was added to each sample and incubated at 37°C overnight. The next day, phenol:chloroform:isoamyl alcohol (25:24:1, v/v; ThermoFisher Scientific 15593049) was used to extract nucleic acids. DNA was precipitated with 100% ethanol (Millipore-Sigma E7023) and dissolved in 25 μl RNase-free water. To remove RNA contamination prior to library preparation, DNA samples were incubated with 25 µg/ml RNase A (ThermoFisher Scientific EN0531) at 37°C for 10 min. Library DNAs were amplified by PCR using Illumina universal i5 primers, Nextera barcoded i7 primers, and NEBNext HiFi 2x PCR Master mix (New England Biolabs M0541). The PCR conditions were: cycle 1: 72°C for 5 min; cycle 2: 98°C for 30 sec; cycle 3: 98°C for 10 sec; cycle 4: 63°C for 10 sec, repeating 14 times, followed by 72°C for 1 min and hold at 8°C. Post-PCR clean-up was performed using 1:1 volume Ampure XP beads (Beckman Coulter A63880). Samples were dissolved in 10 mM Tris-HCl (pH 8; Millipore-Sigma T2694) for sequencing. Size distribution and concentration of libraries was determined by capillary electrophoresis using the 4200 TapeStation (Agilent). Paired-end sequencing (2x150 bp) was performed on pooled-libraries, with 10-12 million reads per library, using the Illumina HiSeq system.