GSM7324994: 4Cseq_501mel shLuc 6D ULK4_int31 rep2; Homo sapiens; OTHER
In brief, 10 x 106 cells were fixed with 1% formaldehyde for 10 min. Cross-linking was quenched with 200 mM glycine and cells were lysed with NP-40 lysis buffer [10 mM Tris-HCl (pH 8.0), 10 mM NaCl, 0.2% NP-40 and 1x complete EDTA-free protease inhibitors (Roche)] for 20 min on ice. Nuclei were incubated with 0.5 % SDS at 65 °C for 10 min followed by 1.25 % Triton X-100 at 37 °C for 15 min. Nuclei were digested with 500 U EcoRI (ALKATI viewpoint) or NlaIII (ULK4 intron 31 viewpoint) overnight. qPCR was performed using primers that span restriction enzyme cut site at the bait versus uncut control region to confirm the efficiency of restriction enzyme digestion to over 90%. Digested chromatin was ligated at 16 °C overnight, reverse cross-linked and purified. For efficient PCR amplification and next generation sequencing, a second round of restriction enzyme digestion was performed using 10 µg 3C product and 100 U DpnII (for both ALKATI and ULK4 intron 31 viewpoints) at 37 °C for 5 hr. Sample was ligated overnight at 16 °C, ethanol precipitated at -80 °C and purified with QIAquick PCR purification column. Inverse PCR was performed using 4C_ALKATI_EcoRI or 4C_ULK4int31_NlaIII (reading) and 4C_ALKATI_DpnII or 4C_ULK4int31_DpnII (non-reading) primers with 2x Phusion high-fidelity PCR mix (NEB). Each 4C sample was amplified in 4 PCR reactions, each containing 200 ng of 4C DNA template for 18 cycles. A second round of PCR was performed to add P5 and P7 adapters as well as index sequences for multiplex sequencing. Double-sided size selection was performed using AMPure XP beads (Beckman coulter) with 0.5x and 1x of the PCR volume. Sequencing was performed on Illumina NovaSeq 6000 sequencer PE75. Each sample was sequenced to generate 10 M reads.