title: LaminB1-DamID seq of human:LHCN-M2 Cells-WT1
design: Genomic DNA of stable lines was purified Quick-DNA Miniprep Plus kit (Zymo Research, D4070) with the RNase A digested step included. The RNase treated genomic DNA were then digested with DpnI overnight (at least 12 h) at 37 C, clean up the DpnI-digested DNA with the Qiagen PCR Purification Kit according to the manufacturers instructions. Ligate the DamID adaptors to 750 ng (up to 15 L) DpnI-digested DNA for 2h at 16 C (15 L DpnI-digested DNA, 2 L 10T4 NDA ligase buffer, 0.8 L dsAdR, 1.2 L H2O), followed by incubating at 65 C for 10 mins to inactivate the ligase. Add 19 L of premade TaDa DpnII digestion buffer( 4 L 10 DpnII buffer, 15 L H2O) and 1L of DpnII Enzyme into the mixture, digest the mixture at 37 for 3 h. Add 118 L premade DamID PCR buffer (16 L 10cDNA buffer, 2.5 L DamID PCR primer (50 M), 3.2 L 10 mM dNTPs, 96.3 L) and 2 L of Advantage2 cDNA polymerase enzyme to the DpnII-digested DNA, split the PCR reaction into 440 L reactions, then perform PCR with the programs as followed: 68 C, 10 min, 1 cycle of 94 C for 30 s,  65 C for 5 min,  68 C, 15 min. 3 cycle of 94 C, 30 s, 65 C, 1 min , 68 C, 10 min, 17 cycle of 94 C, 30 s 65 C, 1 min 68 C, 2 min, followed final extension step with 68 C for 5 mins. Purified PCR products with Qiagen PCR Purification Kit according to the manufacturers instructions. 2 L of purified DNA was sonicated with Covaris E220 Evo sonicator to shear DNA from 300 to 500 bp size fragments. Incubated 1 L of AlwI and sheared mixtures at 37C for overnight to remove the DamID adaptors. Cleanup the mixtures with AMPure Beads according to the manufacturers instructions. 500 ng of cleanup DNA in 20 L then were incubated with 2.5 L end-repair enzyme mix (1.14L T4 DNA polymerase (3U/L), 0.23L Klenow fragment (5U/L), 1,14 L T4 polynucleotide kinase) and 7.5 L of end-repair buffer (3 L 10 T4 DNA ligase buffer, 1.2 L 10 mM dNTPs, 3.3 L H2O) at 30 C for 30 mins.  Adenylation of 3 ends were performed by incubating the mixture with 0.75 l of Klenow 35 exo-enzyme at 37 C for 30 min. Followed by incubating 2.5 L of sequencing adaptor and 2.5 L of NEB quick ligase enzyme at 30 C for 30 min. Two rounds of AMPure beads cleanup then were performed according to manufacturers instructions. The purified DNA then were amplified by PCR condition:   98 C, 30 s. 6 cycles of 98 C, 10 s 60 C, 30 s 72 C, 30 s, then final extension at 72 C for 5mins.  Sequencing was performed with an Illumina HiSeq with 150-bp paired-end read runs.