GSM8957107: Stag2KO_CTCF-Nm_rep2; Mus musculus; OTHER
DNA was lysed overnight at 56 degree in a lysis buffer containing: 200mM NaCl, 0.4% SDS, 100mM Tris-HCl pH7.5, 5mM EDTA pH8.0, 200ug/mL proteinase K, precipitated by isopropanol and dissolved in buffer containing 10mM Tris-HCl pH7.5, 0.1mM EDTA Genomic DNA was extracted from G1 arrested cells. 2 to 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: 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.