title: GSM5949083: OCT4 (CUT&RUN hmC-Seal) 2; Mus musculus; MeDIP-Seq
library_construction_protocol: We performed CUT&RUN as described elsewhere (Patty and Hainer, Nat. Protoc. 16:2633-66, 2021) using recombinant Protein A-micrococcal nuclease (pA-MN). Briefly, we extracted nuclei from ~ 4500000 embryonic stem cells using a nuclear extraction buffer and bound to 500 µl pre-washed lectin-coated concanavalin A magnetic beads (Polysciences). Then, we washed beads in binding buffer. We pre-blocked immobilized nuclei with blocking buffer and washed once in wash buffer. Following this, we incubated nuclei in wash buffer containing primary antibody for one hour at 4°C with rotation. We then incubated in wash buffer containing recombinant pA-MN for 30 min at 4°C with rotation. Using ice-water bath, we equilibrated samples to 0°C and added 3 mM CaCl2 to activate pA-MN cleavage. We then performed sub-optimal digestion, at 0°C for 30 min. Following this, we chelated digestion with 2X STOP+ buffer (including 1.5 pg MNase-digested Saccharomyces cerevisiae mononucleosome-sized DNA spike-in control). We released and separated genomic fragments after RNase A treatment and centrifugation. Finally, we digested protein using proteinase K, and DNA was purified using PCl extraction, followed by ethanol precipitation. Library preparation using the NEB Ultra II Library Preparation Kit (New England Biolabs, Cat#: E7645L) was carried out on 30 ng of CUT&RUN DNA, as per the manufacturer's protocol with modifications described here. In brief, after end-repair and A-tailing, adaptor ligated DNA was purified using AMPure XP Beads at a 0.9X ratio and eluted in 11.5 µL of sterile water. We added three spike-in DNA controls to the adapter ligated DNA to assess specific enrichment of modified DNA fragments. Controls consisted of 0.2ng/µl working stocks of unmethylated and methylated Arabidopsis DNA spike-in controls from the Diagenode DNA methylation control package (cat. no. C02040012). They also included the 5hmC spike-in control DNA (amplified from the APC promoter) from the Active Motif Methylated DNA standard kit (Active Motif, cat. no. 55008). We combined 0.3 ng of each spike-in DNA in a final volume of 4.5 µl per experimental sample. We mixed the adapter ligated DNA with the spike-in DNA mix. Then we aliquoted 1.6 µl of this mix into a separate PCR tube and stored it at -20°C, as an input control. We 5hmC glucosylated the remaining 14.4 µl CUT&RUN DNA mixed with spike-in controls, as previously described (Han et al. Mol. Cell 63:711-19, 2016), with the below modifications. Briefly, we 1 µ1 diluted a 3 µM stock of N3-UDP- Glc (aka UDP-Azide-Glucose) from Active Motif (cat. no. 55020) in 1× phosphate-buffered saline (PBS) to establish a working stock of 1.5 µM for Mastermix preparation. We prepared a 20 µl glucosylation Mastermix per experimental sample consisting of the following: 14.4 µl CUT&RUN DNA mixed with spike-in controls, 50 µM HEPES (pH 8.0), 25 mM MgCl2 , 0.1 mM N3-UDP-Glc and 1 U of T4 Phage β-glucosyltransferase (NEB cat. no. M0357L). We incubated the mix for 1 h at 37°C, to promote glucosylation. We then performed biotinylation of azide-labeled 5hmC residues of the glucosylated DNA fragments. We prepared 20mM DBCO-PEG4-Biotin Conjugate (Bioscience cat. no. CLK-A105P4-10) in sterile water and stored it (at -20°C), in one-time use aliquots to avoid freeze-thaw. We mixed 20 µl of glucosylated DNA with 1.8 µM DBCO-PEG4-Biotin in a final reaction volume of 22 µl, then incubated 2 h at 37°C to promote biotinylation. Then, we prepared BioRad MicroSpin P-30 Gel Columns (cat. no. 7326223), per manufacturer's protocol, and used them to purify total DNA fragments from reaction components. Briefly, we loaded the sample onto the column, then centrifuged 4 min at 1000 × g to elute purified DNA sample in 22 µl of Tris buffer. To specifically capture biotinylated 5hmC DNA fragments, we prepared 2× binding and washing (B&W) buffer (10 mM Tris-HCl, 10 mM EDTA, 2 M NaCl). Using 20 µg of MyOne Streptavidin C1 Dynabeads (ThermoFisher, cat. no. 65001), we re-suspended in 0.2 ml of 1× B&W buffer per experimental sample to wash beads. We subjected beads to 3 total washes, then re-suspended to a final volume of 22 µl per sample in 2× B&W buffer. We added 22 µl of purified total DNA fragments to 22 µl of washed beads, then incubated 15 min under gentle rotation to promote streptavidin-biotin binding. To isolate beads containing streptavidin-bound biotinylated DNA fragments, we incubated them on magnet for 3 min. Then, we washed the beads 3 times with 1× B&W buffer to remove non-biotinylated DNA fragments lacking 5hmC. Beads were re-suspended in 50 µl of low TE buffer. We conducted reverse transcriptase qPCR (RT-qPCR) to compare enrichment of 5hmCspike- in after biotin enrichment relative to input sample stored earlier. RT-qPCR in the form of a 10µl reaction, consisted of 1× KAPA SYBR Fast qPCR Mastermix (KAPA, cat. no. KK4601), 1 µl of template DNA, and primers at a final concentration of 0.3 µM. We set up different reactions for each primer set to detect each spike-in control DNA separately. We used template-specific forward and reverse primers to detect the 5hmCspike-in DNA fragment from the Active Motif Methylated DNA standard kit, or the methylated- or unmethylated- Arabidopsis DNA spike-in controls from the Diagenode DNA Methylation Control package kit. We amplified with the following PCR program: 98°C for 30 s, followed by 40 cycles of 98°C for 15 s and 60°C for 15 s (with image capture), ending with melt curve analysis. To generate bead-free template for library DNA amplification, we established a PCR reaction mix containing 0.3 µM of unique dual index primers (NEB cat. no. E6440S), 1× NEBUltra II Q5 MM, and DNA/bead template for a final volume of 100 µl per sample. We split samples into 2× 50 µl reactions and amplified using the following PCR program: 98°C for 30 s, followed by 5 cycles of 98°C for 10s, 60°C for 75s, ending with a hold at 4°C. We then transferred reaction tubes to a magnetic rack and transferred bead-free supernatant to new PCR tubes. To amplify DNA libraries for a maximum of 16 cycles total (including initial 5 cycles), we used the same PCR conditions for the bead-free template. We then dual size selected DNA libraries using AMPure XP beads at 0.7× to 1.0× ratio.