HiSeq X Ten paired end sequencing; 4C-Seq identifies the chromatin interaction landscape of the miR-146a promoter region
4C-Seq identifies the chromatin interaction landscape of the miR-146a promoter region
1x10^7 cells were crosslinked in 1% formaldehyde for 10 min at room temperature. After crosslinking, cells were quenched with 125 mM Glycine solution. Cells were collected and incubated in lysis buffer while tumbling for 10 min at 4°C. After lysis, the pellet of nuclei was washed and resuspended in Csp6I buffer. 0.3% SDS was added and incubated at 37°C in a shaker at 750 rpm for 1 hour. Then, 2% Triton X-100 was added and cells were incubated for another hour to sequester the SDS. 400 units of  MboI enzyme  were added for a 4 hours incubation at 37 °C in a shaker at 900 rpm; another 400 units of MboI were added for overnight incubation. 1.5% SDS was added and incubated for 20 min at 65°C to inactivate MboI. Cells were suspended with ligation buffer and 1% Triton X-100 to a volume of 7 mL and then incubated for 1 h at 37 °C with occasional shaking. 100 units of T4 DNA ligase (M0202S, NEB) were added and incubated for 4 h at 16°C, followed by 30 min at 25°C. After that, 300 mg Proteinase K was added for overnight incubation at 65°C and 30 μl of RNaseA solution was added and incubated for 45 min at 37°C. DNA was purified using phenol-chloroform and dissolved in restriction buffer with 50 units of NlaIII (R0125L, NEB) and incubated for overnight in a shaker at 37°C and 900 rpm, followed by treatment with 65°C for 25 min to heat inactivation of the enzyme. The sample was diluted with ligation buffer to a final volume of 14 mL, 100 units of ligase were then added for incubation for 4 h at 16 °C, followed by 30 min at 25 °C. DNA was purified using phenol-chloroform and further purified with QIAquick PCR purification kit (28106, Qiagen). The DNA concentration was detected by Qubit (ThermoFisher). 1ug of each template was amplified  to construct the 4C-seq library with locus-specific primers containing Illumina sequences. The libraries were purified and sequencing was performed on a HiSeq x ten (Illumina) DNA was purified using phenol-chloroform and further purified with QIAquick PCR purification kit (28106, Qiagen). The DNA concentration was detected by Qubit (ThermoFisher) 1ug of each template was amplified  to construct the 4C-seq library with locus-specific primers containing Illumina sequences.