GSM5241375: ESC_DUX_Clone1_RPA_ChIP; Mus musculus; ChIP-Seq
Twenty million of untreated or DOX-treated ESCDUX cells were fixed using 1% Formaldehyde (Sigma, F1635) at 37°C for 10 min. Fixation was then quenched with 125mM glycine (Sigma). Cell pellets were washed twice with cold PBS and samples were snap-frozen and stored in −80°C. Frozen pellets were resuspended in 1mL RIPA buffer (10 mM Tris-HCl pH 7.5, 1 mM ethylenediaminetetraacetic acid (EDTA), 0.1% sodium dodecyl sulphate, 0.1% sodium deoxycholate, 1% Triton X-100, and 1 Complete Mini EDTA-free proteinase inhibitor tablet (Roche)). Sonication was performed using Covaris S220 (duty cycle 20%, peak incident power 175, and cycle/burst 200 for 30 min at 4 °C). Chromatin was pre-clarified with 40 μL prewashed Dynabeads Protein A (ThermoFisher) for 30 min at 4 °C and then incubated with 40 μL Dynabeads Protein A bound to 10mg of anti-RPA32 antibody (Abcam ab10359) or 10mg of Guinea Pig anti-Rabbit IgG (ABIN101961, Antibodies-online) in 100uL PBS overnight at 4°C. Beads were then collected in a magnetic separator (DynaMag-2 Invitrogen), washed twice with cold RIPA buffer, twice with RIPA buffer containing 0.3M NaCl, twice with LiCl buffer (0.25 M LiCl, 0.5% Igepal-630, 0.5% sodium deoxycholate), once with TE (10 mM Tris pH 8.0, 1mM EDTA) + 0.2% Triton X-100, and once with TE. Crosslinking was reversed by incubating the chromatin bound beads at 65°C for 4 hours in the presence of 0.3% SDS and 1mg/mL of Proteinase K (Qiagen). The entire ChIP DNA was used to prepare Illumina sequencing libraries. End-repair was performed in 75 μl of T4 ligase reaction buffer, 0.4 mM of dNTPs, 4 U of T4 DNA polymerase (NEB), 13.5 U of T4 Polynucleotide Kinase (NEB) and 1.5 U of Klenow fragment (NEB) at 24°C for 30 min in a ThermoMixer C at 400 rpm. End-repair reaction was cleaned using 2X Agencourt AMPure XP beads and eluted in 15 μl of EB that was used for A-tailing reaction in 30 μl of NEBNext dA-Tailing reaction buffer (NEB) with 7.5 U of Klenow fragment exo- (NEB) at 37°C for 30 min. The 30 μl of the A-tailing reaction were mixed with Quick Ligase buffer 2X (NEB), 3,000 U of Quick ligase and 5 nM of annealed adaptor (Illumina truncated adaptor) in a volume of 75 μl and incubated at 25C for 20 min. Adaptor was prepared by annealing the following HPLC oligos: 5'-phosphate/GATCGGAAGAGCACACGTCT-3' and 5'-ACACTCTTTCCCTACACGACGCT CTTCCGATC∗T-3' (∗phosphorothioate bond). Ligation was stopped by adding 50mM of EDTA and cleaned with 1.8X Agencourt AM- Pure XP beads and eluted in 15ul of EB that was used for PCR amplification in a 50 uL reaction with 1 μM primers TruSeq barcoded primer p5, AATGATACGGCGACCACCGAGATCTACACNNNNNNNNACACTCTTTCCCTACACGACGCTCTTCCGATC∗T, TruSeq barcoded primer p7, CAAGCAGAAGACGGCATACGAGANNNNNNNGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC∗T, (NNNNNNNN represents barcode and ∗ a phosphothiorate bond), 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 Agencourt AMPure XP beads (Beckman Coulter), run on a 2% agarose gel and a smear of 200-500bp 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 Nextseq500 (75bp single-end reads).