GSM5208207: mESC_CUT&RUN_DMSO_H3K27ac_rep2; Mus musculus; OTHER
The CUT&RUN protocol was carried out as previously described (Skene et al., 2018) with modifications. Cells were harvested via gentle agitation post the incubation with Accutase for 5 minutes and collected via centrifugation at 300 g for 3 minutes at 4°C. The cells were washed twice with ice-cold PBS and nuclei were isolated with Nuclear extraction buffer [20 mM HEPES-KOH, pH 7.9, 10 mM KCl, 0.5 mM Spermidine (Sigma #05292-1ml-F), 0.1% Triton X-100, 20% Glycerol and 1x protease and phosphatase inhibitor (ThermoFisher #78442)]. To aid in nuclear capture, isolated nuclei were incubated with activated Concanavalin A beads (Bangs Laboratories #Bp531) for 15 minutes at room temperature followed by incubation with the desired primary antibody (1:100 dilution) overnight. Any unbound antibody was washed away using Wash Buffer (20 mM HEPES-NaOH pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, 1x protease phosphatase inhibitor) for a total of two washes. Supernatant was discarded via bead liquid separation using a magnetic stand and pA-MNase was added at a final concentration of 700 ng/ml (Batch 6: Stock concentration: 143 μg/ml) and the complex was incubated for 1 hour at 4°C. Following incubation, the bound complexes were washed twice with Wash Buffer and placed in a 0°C metal block for 5 minutes to pre-chill the tubes. To activate pA-MNase, CaCl2 was added to the pre-chilled bound complexes at a final concentration of 2 mM and incubated in the 0°C metal block for 30 minutes. The digestion reaction was neutralized by the addition of an equal volume of 2x STOP buffer (200 mM NaCl, 20 mM EDTA, 4 mM EGTA, 50 μg/mL RNase A, 40 μg/mL glycogen and 100 pg/mL heterologous spike in DNA) followed by incubation at 37°C for 10 minutes to released cleaved fragments of DNA. The targeted protein-DNA complex was released via centrifugation at 16000 g for 5 minutes and supernatant was collected and transferred to freshly labelled 1.7 mL tubes. For H3K27ac, a spin column method (Macherey-Nagel #740609.250) of extraction was followed according to the manufacturer's instructions to recover large DNA-protein complexes. To enrich for smaller fragments occupied by non-histone proteins (BRD4, NIPBL and RAD21), a phenol chloroform extraction protocol was followed as briefly described. The supernatant was decanted into a fresh tube to which 10% SDS (final concentration 0.1%) and proteinase K (final concentration of 20 mg/ml) was added and mixed by inversion followed by incubation for 10 minutes at 70°C. Post incubation, phenol-chloroform-isoamyl alcohol was added to the solution and vortexed at full speed to mix. The solution was transferred to a phase lock tube, centrifuged at 16000 g for 5 minutes and inverted 10 times post the addition of chloroform followed by another round of centrifugation at 16000 g for 5 minutes. The supernatant was transferred to a fresh tube containing glycogen (final concentration 20 µg/mL) and DNA was precipitated via the addition of 100% ethanol. The samples were incubated on ice for 10 minutes and subsequently centrifuged at 16000 g at 4°C. Excess liquid was carefully discarded and 1 ml of ethanol was added to the pellet and tubes were mixed by inverting. Samples were centrifuged once again at 16000 g at 4°C. The supernatant was carefully discarded, and the pellet was air dried for 10 minutes and resuspended in a desired amount of TE buffer (1 mM Tris-HCL pH 8, 0.1 mM EDTA). Construction of the CUT&RUN DNA libraries for next generation sequencing was carried out with modifications using the KAPA Hyper Prep Kit (KAPA Biosystems #KK8502 07962347001), coupled with the NEBNext Multiplex Oligos for Illumina (New England Biolabs #E6440S). Briefly, the end repair and A-tailing temperature was dropped from the recommended 65°C to 58°C to avoid denaturation of small AT rich fragments and the reaction time for this step was increased to 60 minutes to compensate for lower enzymatic activity. Adapter ligation was performed using the NEB Adapter stock diluted down to the desired concentration. Post adapter ligation, the recommended volume of NEB USER enzyme was added to the reaction and incubated 37°C for 15 minutes ensuring digestion of the uracil bridge allowing successful pairing of the primers containing barcodes in the successive steps of the reaction. After adapter ligation and hairpin cleavage, 1.1x AgenCourt AMpure Beads (Beckman Coulter #A63880) were added to the reaction to ensure high recovery of short to medium sized fragments. For the amplification reaction, 10µl of the NEBNext Multiplex Oligos were added to the samples, replacing the KAPA Library Amplification Primer Mix (10X) and subjected to 14 cycles of amplification. Post amplification cleanup was carried out using 1.1x AgenCourt AMpure Beads. The libraries were quantified via qPCR using the KAPA Library Quantification Kit for Illumina Platforms (KAPA Biosystems #KK4835 07960204001) and quality control was carried out on the Agilent BioAnalyzer 2100 Expert (Agilent Technologies, USA) using the High Sensitivity DNA Kit (Agilent Technologies #5067-4626). 40 barcoded libraries were quantified and pooled together in equimolar ratio with a total of 20 libraries per pool. The pooled libraries were denatured according to the standard Illumina protocol.