GSM8574665: UMI4C_PF-382_IRX3_CTCF3pbait_techrep1; Homo sapiens; OTHER
5 µg of 3C template from each experimental condition was sonicated at 4ºC for 20 cycles (30s ON/ 60 s OFF) on a Bioruptor Pi.co (Diagenode). The post-sonication size distribution was checked on a 2200 Tapestation (Agilent) with D1000 Screentape to ensure an ideal peak distribution size of 450 – 550 bp. The sonicated DNA was end-repaired by the End Repair Module (NEB – E6050L) and cleaned up with 2.2x AmpureXP beads (Beckman Coulter) followed by dA-tailing (NEB – E6053L) as per the manufacturers' reaction conditions and instructions. Following A-tailing, DNA was cleaned up with 2x AmpureXP beads and eluted in 67 µL of EB buffer (Qiagen). Illumina compatible adapters (IDT – xGEN UDI-UMI adapters) were ligated by using T4 ligase (NEB – M0202S) overnight at 16ºC. Two nested PCR reactions were conducted with the following bait sequences for the FTO intron 8 CTCF site, 5'-GAAAGGCTTGTTCTTCCTGGC-3' for bait 1 (upstream primer), 5'- AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTTGGAACTGCTGTTTCCCACTT-3' for bait 2 (Illumina prefix appended to downstream primer). The bait sequences for the IRX3 proximal promoter were, 5'-AGGAAGAAGTGAAGGGAACGGA-3' for bait 1 (upstream primer), 5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTTGCAGGAGCCCGAAGCA-3' for bait 2 (Illumina prefix appended to downstream primer). The bait sequences for the CTCF site 5' to IRX3 were, 5'-GAGAACCTTCCTTGCGCGAC-3' for bait 1 (upstream primer), 5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTCCGGCGCTAACCTCTTTTCT-3' for bait 2 (Illumina prefix appended to downstream primer). The bait sequences for the CTCF site 3' to IRX3 were, 5'-TCAACACTGCACACCTTCGA-3' for bait 1 (upstream primer), 5-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTGGGAGTTCCCTCCGCGAA-3' for bait 2 (Illumina prefix appended to downstream primer) and 5'-CAAGCAGAAGACGGCATACGA-3' for the Illumina enrichment primer. The PCR reactions were performed in 50 μl volume with the following reagents: 10 μl GoTaq Flexi buffer (Promga M792A), 3 μl MgCl 25 mM (Promega A3511), 0.2 mM dNTPs, 2 μl 10 mM bait 1/upstream primer (0.4 mM final concentration), 2 μl 10 mM Illumina enrichment primer (0.4 mM final concentration), 1 μl GoTaq Hot Start (Promega M5005) and 200 ng UMI-4C DNA template. PCR program: 2 min 95 °C, 20 cycles of 30 s 95 °C, 30 s 56 °C and 60 s 72 °C and final extension of 5 min 72 °C. Amplified DNA was cleaned with 1x AmpureXP beads, and the eluate was used as a template in the second PCR reaction. Conditions for the second PCR reaction were identical to the first, and the only differences were the use of the bait primer 2. The molarity of the libraries were quantified by using a Qubit dsDNA HS Assay Kit (ThermoFisher – Q32851) together with a Tapestation (Agilent). Libraries were pooled and diluted to 4 nM concentration and multiplexed with other libraries, including a 10% PhiX spike-in (Illumina) and sequenced on a MiSeq (Illumina) with a MiSeq Reagent Nano Kit v2, 300 cycles (Illumina).