title: Illumina HiSeq 2500 sequencing; 4C-seq from viewpoint at MYC promoter (VP-MYC1 and VP-MYC2), in wild type (Hap) and variously modified alleles around the locus in human iPS cells.
design: 4C-seq from viewpoint at MYC promoter (VP-MYC1 and VP-MYC2), in wild type (Hap) and variously modified alleles around the locus in human iPS cells.
library_construction_protocol: The cells were dissociated from dish with TrypLE™ Select (Thermo Fisher Scientific K.K., Cat#12563-011) . We cultured the cells in the StemFit® AK02N medium (ReproCELL, Cat#RCAK02N) on dish coated with iMatrix-511 (ReproCELL, Cat#NP892-012) without feeder cells. DOX (Sigma, D9891) was basically added at the final concentration of 10 ng/ml, unless specifically indicated. When DOX was removed for time-course analysis, the concentration was first changed to 1 ng/ml one day before the start of removal. Then at the start of the removal, the cells were first washed with PBS (Thermo Fisher Scientific K.K., 10010-049) and then fresh medium without DOX was supplied. Further two hours later, wash with PBS and replacement of medium was repeated to ensure the removal of DOX.  We fixed the cells in 2% paraformaldehyde for 10 minutes at room temperature. Then the cells were lysed in lysis buffer (50 mM Tris (pH7.5), 150 mM NaCl, 5 mM EDTA, 0.5% NP-40, 1% Triton X-100, 1x complete proteinase inhibitors (Roche, Cat#11697498001); 1 ml), passed through a 23-guage needle, pelleted and frozen in liquid nitrogen. After the cells were resuspended in H2O and CutSmart® Buffer (New England Biolabs, Cat#B7204) and treated with 0.3% SDS and 2.5% Triton X100 at 37°C for 1 hour, respectively, we performed first digestion of the chromatin with 25 units of NlaIII restriction enzyme (New England Biolabs, Cat#R0125) on a rotator at 37°C for overnight. After heat inactivation of the enzyme, 12.5 units of T4 DNA ligase (Thermo Fisher Scientific, Cat#EL0014) were applied for self-ligation of the digested chromatin. After de-crosslinking and purification, we carried out second digestion with 20 units of DpnII restriction enzyme (New England Biolabs, Cat#R0543). Then the chromatin was again self-ligated with 12.5 units of T4 DNA ligase (Thermo Fisher Scientific, Cat#EL0014). We then performed the inverse PCR from the chromatin as the template to amplify the 4C library from a given viewpoint for 25 cycles with Tks Gflex™ DNA Polymerase (Takara, Cat#R060A). The primer sequences for VP-MYC1 were 5'-TCTTTCCCTACACGACGCTCTTCCGATCTGTAGGCGCGCGTAGTTAATTC-3' and 5'-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTCGCTAAGGCTGGGGAAAG-3'; those for VP-MYC2 were 5'-TCTTTCCCTACACGACGCTCTTCCGATCTTCTCCCTGGGACTCTTGAT-3' and 5'-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTAAACTCCCATTGCATTTGTTG-3'. We purified the DNA with High-pure PCR Product Purification Kit (Roche, Cat#11732676001) and performed the 2nd round of PCR to attach to the libraries adaptor and index sequences for the NGS analysis for 8 cycles again with Tks Gflex™ DNA Polymerase (Takara, Cat#R060A). The DNA was purified with High-pure PCR Product Purification Kit (Roche, Cat#11732676001).