GSM8559297: eHAP1, C17_PDS5A-AID, TACL-ON_IAA, rep1; Homo sapiens; OTHER
4C templates preparation was performed as described in PMID: 31351925. In brief, ten million cells per sample were crosslinked with 2% formaldehyde, followed by quenching by glycine at final concentration of 0.125 M. Four-cutter restriction enzyme MboI (New England BioLabs) was used for in situ digestion (300U/10 million cells). Digested DNA fragments were ligated, reverse-crosslinked and subsequently purified through isopropanol and magnetic beads (Macherey-Nagel NucleoMag PCR Beads). Four-cutter restriction enzyme Csp6I (CviQI, ThermoFisher ER0211, 50U/sample) was used for template trimming. Indexed Illumina sequencing adapters were introduced to ligation fragments of interest with a two-step PCR strategy using ELT PCR System (Roche, cat. no. 11681842001). To prevent PCR amplification and sequencing of TetO repeats due to tandem ligation of two or more TetO DpnII fragments in a given 4C circle, an in vitro digestion of 4C templates was performed as in PMID: 31932773, with the following modifications: two sgRNA were used to target Cas9 into the TetO repeats between viewpoint primers; pre-incubation of Cas9 protein and sgRNA template at room temperature. In brief, two sgRNA templates were obtained using the Megashortscript T7 transcription kit (Invitrogen) followed by 4x AMPure XP (Agencourt) purification. Purified Cas9 protein (generated by Hubrecht protein facility) was pre-incubated with the sgRNAs for 30min at room temperature. 4C templates were subsequently added to the pre-incubated Cas9/sgRNA complexed for overnight digestion at 37°C. Cas9 protein was inactivated by incubating at 70 °C for 5 min. Resulting products were purified with 1x AMPure XP and used as PCR template for TetO dedicated 4C.