title: GSM5027349: ESC_DUX_Clone 1_CTCF; Mus musculus; OTHER
library_construction_protocol: The CUT&RUN protocol was slightly modified from (Skene et al., 2018; Meers et al., 2019). In brief, trypsinized or cell sorted ESC (between 150,000-500,000 cells depending on the experiment) were washed three times with Wash Buffer (20 mM HEPES-KOH pH 7.5, 150 mM NaCl, 0.5 mM spermidine, Roche complete Protease Inhibitor tablet EDTA free) and bound to activated Concanavalin A beads (Polysciences) for 10 minutes at room temperature. Cells were then permeabilized in Digitonin Buffer (0.05 % Digitonin and 0.1% BSA in Wash Buffer) and incubated with the antibody against CTCF (07-729, Millipore) at 4°C for 2 hours. For negative controls, Guinea Pig anti-Rabbit IgG (ABIN101961, Antibodies-online) was used. Cells were washed with Digitonin Buffer following antibody incubation, and further incubated with purified hybrid protein A-protein G-Micrococcal nuclease (pAG-MNase) at 4°C for 1 hour. Samples were washed in Digitonin Buffer, resuspended in 150 μl Digitonin Buffer and equilibrated to 0°C on ice water for 5 minutes. To initiate MNase cleavage, 3 μl 100 mM CaCl2 was added to cells and after 1 hour of digestion, reactions were stopped with the addition of 150 μl 2x Stop Buffer (340 mM NaCl, 20 mM EDTA, 4 mM EGTA, 0.02 % Digitonin, 50 μg/ml RNase A, 50 μg/ml Glycogen). Samples were incubated at 37°C for 10 minutes to release DNA fragments and centrifuged at 16,000 g for 5 minutes. Supernatants were collected and a mix of 1.5 μl 20% SDS / 2.25 μl 20 mg/ml Proteinase K was added to each sample and incubated at 65°C for 35 minutes. DNA was precipitated with ethanol and sodium acetate and pelleted by high-speed centrifugation at 4°C, washed, air-dried and resuspended in 10 μ 0.1x TE. The entire precipitated DNA obtained from CUT&RUN was used to prepare Illumina compatible sequencing libraries. In brief, end-repair was performed in 50 μl of T4 ligase reaction buffer, 0.4 mM dNTPs, 3 U of T4 DNA polymerase (NEB), 9 U of T4 Polynucleotide Kinase (NEB) and 1 U of Klenow fragment (NEB) at 20°C for 30 minutes. End-repair reaction was cleaned using AMPure XP beads (Beckman Coulter) and eluted in 16.5 μl of Elution Buffer (10 mM Tris-HCl pH 8.5) followed by A-tailing reaction in 20 μl of dA-Tailing reaction buffer (NEB) with 2.5 U of Klenow fragment exo- (NEB) at 37°C for 30 minutes. The 20 μl of the A-tailing reaction were mixed with Quick Ligase buffer 2X (NEB), 3000 U of Quick Ligase (NEB) and 10 nM of annealed adaptor (Illumina truncated adaptor) in a volume of 50 μl and incubated at room temperature for 20 min. The adaptor was prepared by annealing the following HPLC-purified oligos: 5′-Phos/GATCGGAAGAGCACACGTCT-3′ and 5′-ACACTCTTTCCCTACACGACGCTCTTCCGATC∗T-3′ (∗phosphorothioate bond). Ligation was stopped by adding 50 mM of EDTA, cleaned with AMPure XP beads and eluted in 14 μl of Elution Buffer. All volume was used for PCR amplification in a 50 μl reaction with 1 μM primers TruSeq barcoded primer p7, 5′-CAAGCAGAAGACGGCATACGAGATXXXXXXXXGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC∗T-3′ and TruSeq barcoded primer p5 5′-AATGATACGGCGACCACCGAGATCTACACXXXXXXXXACACTCTTTCCCTACACGACGCTCTTCCGATC*T-3′ (∗ represents a phosphothiorate bond and XXXXXXXX a barcode index sequence), and 2X Kapa HiFi HotStart Ready mix (Kapa Biosciences). The temperature settings during the PCR amplification were 45 s at 98°C followed by 15 cycles of 15 s at 98°C, 30 s at 63°C, 30 s at 72°C and a final 5 min extension at 72°C. PCR reactions were cleaned with AMPure XP beads (Beckman Coulter), run on a 2% agarose gel and a band of 300bp approximately was cut and gel purified using QIAquick Gel Extraction Kit (QIAGEN). Library concentration was determined with KAPA Library Quantification Kit for Illumina Platforms (Kapa Biosystems). Sequencing was performed on the Illumina NextSeq550 (75bp pair-end reads).