title: GSM7136463: 4C_B21_WT_for_f_del_rep1 [QHR4C-seq]; Mus musculus; OTHER
library_construction_protocol: 1 × 10^6 of cortex neurons or cultured cell were used for one experiment. Neuron cells were first dissected and digested using 0.00625% collagenase. Collagenase was quenched with 2-fold volumes of 10%FBS in NeuroBasal Medium and the suspended cells were used for library construction. The neuron suspension or cultured cell were cross-linked using 2% formaldehyde in 10% FBS/PBS for 10 min at room temperature followed by washing twice with PBS solution containing proteinase inhibitors (Roche). Cells were then resuspended and lysed twice with 1ml prechilled lysis buffer containing proteinase inhibitors (Roche) for 10 min with slow rotations. The cells were then digested overnight at 37 °C with DpnII while shaking at 900 rpm. After inactivation of DpnII at 65 °C for 20 min, proximity ligation was carried out for 24 hr at 16 °C with T4 DNA ligase in 0.1 ml 1 × T4 ligation buffer. The ligated product was then reverse cross-linked and DNA was extracted using phenol-chloroform. Glycogen was added to facilitate DNA precipitation. The precipitated DNA was then dissolved in 50 ml water, and sonicated using the Biorupter system (with low energy setting at a train of 30-second sonication with 30 second intervals for 12 cycles) to obtain DNA fragments ranging from 200 to 600 bp.The captured fragments were linear-amplified using a 5' biotin-tagged primer complementary to the viewpoint fragment in 100ul of PCR system for a total of 60 cycles. This primer should be neither too close to the DpnII site to facilitate the nested PCR at the final amplification step, nor too far away from the DpnII site to maximize the product amount. The amplification products were denatured by incubating at 95 °C for 5 min, and immediately chilled on ice to obtain ssDNA. The ssDNA was then enriched and purified with Streptavidin Magnetic Beads (Invitrogen) according to the manufacturer's instructions. The ssDNA on-beads was then ligated in 15 µl with 0.1 µM of adapters (Supplementary Table 1) at 16 °C for 24 hr. The adapters were generated by annealing two complementary primers matching Illumina P7 sequences. After ligation, free adapters were removed by washing the beads twice with the B/W Buffer (5 mM Tris-HCl, 1 M NaCl, 0.5 mM EDTA, pH 7.5). Finally, the QHR-4C libraries were generated by one-step PCR amplification (94 °C, 2 min; 94 °C, 10 sec, 60 °C, 15 sec, 72 °C, 1 min for 19 cycles; and a final extension at 72 °C, 5 min) with captured ssDNA on beads as the template and a pair of PCR primers. The forward primer matches the Illumina P5 and the viewpoint sequence adjacent to the viewpoint DpnII site with barcodes, and the reverse primer matches Illumina P7 with indexes. The PCR products were purified with a PCR purification kit (Qiagen).