title: GSM8755774: 3C_Wapl-AID2_CTCF-Nm_5PhIAA_rep2; Mus musculus; OTHER
library_construction_protocol: 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 degrees celsius, 1 hour) and 1.8% Triton X-100 treatment (37 degrees celsius, 1 hour) successively, followed by overnight NalIII (700 units) digestion at 37 degrees celsius.  DpnII was inactivated at 65 degrees celsius 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 degrees celsius, ON) and treated with RNaseA (37 degrees celsius, 1 hour). DNA was purified via phenol/chloroform extraction and resuspended in 200ul 1XTE buffer. 4 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: 98 degrees celsius 2min, 1cycle; 95 degrees celsius 30sec, 58 degrees celsius 30sec, 72 degrees celsius 90sec, 80 cycles; 72 degrees celsius 2min, 1 cycle). Single-stranded PCR products were purified via Dynabeads MyOne streptavidin C1 beads and ligated to bridge adaptor (25 degrees celsius 1 hour, 22 degrees celsius 3 hours, 16 degrees celsius overnight). Adaptor-ligated Products were amplified via nested PCR with indexed locus-specific primers and adaptor primer (PCR with Phusion enzyme: 95 degrees celsius 5min, 1cycle; 95 degrees celsius 30sec, 60 degrees celsius 30sec, 72 degrees celsius 60sec, 15 cycles; 72 degrees celsius 6 min, 1 cycle). The PCR products were further tagged with P5-I5 and P7-I7 sequences (95 degrees celsius 5min, 1cycle; 95 degrees celsius 30sec, 60 degrees celsius 30sec, 72 degrees celsius 60sec, 12 cycles; 72 degrees celsius 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.