GSM8957038: 3C_WT_rep5; Mus musculus; OTHER
10 million cells were cross-linked with 2% formaldehyde at room temperature for 10 minutes, quenched by glycine (final 0.125M), and then lysed with cold cell lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.5% NP-40, 1% Triton X-100, one tablet of protease inhibitor in 50 ml) on ice for 10 min. Nuclei were subjected to 0.3% SDS treatment (37 o, 1 hour) and 1.8% Triton X-100 treatment (37 o, 1 hour) successively, followed by overnight NalIII (700 units) digestion at 37 o .  DpnII was inactivated at 65 o for 20 min and samples were subjected to ligation under dilute condition with T4 DNA ligase (100 units). Ligated chromatin was de-crosslinked with Proteinase K (56 o, ON) and treated with RNaseA (37 o, 1 hour). DNA was purified via phenol/chloroform extraction and resuspended in 200ul 1XTE buffer. 10 ug of DNA were sonicated via Diagenode bioruptor (20 sec on, 1 min off, 2 cycles with low energy input) and subjected to linear amplification with a biotinylated primer (PCR with Phusion enzyme: 98o 2min, 1cycle; 95o 30sec, 58o 30sec, 72o 90sec, 80 cycles; 72o 2min, 1 cycle). Single-stranded PCR products were purified via Dynabeads MyOne streptavidin C1 beads and ligated to bridge adaptor (25o 1 hour, 22 o 3 hours, 16 o overnight). Adaptor-ligated Products were amplified via nested PCR with indexed locus-specific primers and adaptor primer (PCR with Phusion enzyme: 95o 5min, 1cycle; 95o 30sec, 60o 30sec, 72o 60sec, 15 cycles; 72o 6 min, 1 cycle). The PCR products were further tagged with P5-I5 and P7-I7 sequences (95o 5min, 1cycle; 95o 30sec, 60o 30sec, 72o 60sec, 12 cycles; 72o 6 min, 1 cycle), size-selected via gel extraction (500-1000 bp) and loaded to Mi-Seq machine (Illumina) with paired-end 300 bp sequencing.