title: Illumina HiSeq 2500 sequencing; 4C-seq to show the effects of insertion of STITCH into MYC+30kb and NEUROG2-65kb positions on the chromatin conformation in human iPSCs and differentiated neural progenitor cells
design: 4C-seq to show the effects of insertion of STITCH into MYC+30kb and NEUROG2-65kb positions on the chromatin conformation in human iPSCs and differentiated neural progenitor cells
library_construction_protocol: The cells were dissociated from dish with TrypLE™ Select (Thermo Fisher Scientific K.K., Cat#12563-011) . coated with iMatrix-511 (ReproCELL, Cat#NP892-012) without feeder cells.  DOX (Sigma, D9891) was added at the final concentration of 10 ng/ml. To differentiate the iPSCs to NPCs, when the iPSCs became almost confluent, the medium was switched to the neural induction medium consisting of 1:1 of DMEM/Ham's F-12 (FUJIFILM Wako, Cat#042-30795) and Neurobasal Plus Medium (Thermo Fisher Scientific K.K., Cat#A3582901), 1X GlutaMAX Supplement (Thermo Fisher Scientific K.K., Cat#35050061), 1X MEM Non-Essential Amino Acids Solution (Thermo Fisher Scientific K.K., Cat#11140050), 1X Penicillin-Streptomycin (Thermo Fisher Scientific K.K., Cat#15140122), 1X N-2 Supplement (Thermo Fisher Scientific K.K., Cat#17502048), 1X B-27 supplement (Thermo Fisher Scientific K.K., Cat#17504044), 0.1 mM 2-Mercaptoethanol (Sigma, Cat#M7522), 250 nM LDN-193189 (StemRD) and 10 μM SB-431542 (Tocris, Cat#1614). The medium was changed every or every other day up to day 6. 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). 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).