GSM5856754: RAW_4C_seq_silencer_rp1; Mus musculus; OTHER
10^7 RAW 264.7 cells were re-suspended with 1ml lysis buffer for 10 minutes. Nuclei were re-suspended into 440 ul Milli-Q water and 60 ul of digestion buffer was added. The cell lysis mixture was then incubated at 37C for 2 hours in the shaker (900 rpm). 200U DpnII was added into the mixture three times. The lysis mixture was then incubated in the shaker overnight. The reaction mix was inactivated after overnight digestion by incubating at 65C for 20 min. The samples were then transferred to 50 ml Falcon tubes with 700 ul ligation buffer and 5.5 ml Milli-Q water. The samples were then supplemented with 100 U T4 ligase and incubated overnight at room temperature. After that, samples were de-crosslinked at 65C for 12 hours. The ligated DNA was then purified by the phenol-chloroform method, and further digested with NlaIII at 37C overnight. The restriction enzyme was then inactivated for 20 min at 65C. The DNA was further ligated at room temperature overnight. After the second ligation, the DNA was purified by QIAquick PCR purification kit. 4C-PCR products DNA was processed using Takara SMRTer ThruPLEX DNA-seq Kit. Briefly, DNA was end-repaired and ligated to stem-loop adaptors. Then DNA was PCR amplified with Illumina-compatible primers for 7-11 cycles and library fragments of 200 to 400 bp were selected using Ampure XP beads.