title: GSM9201364: WT_NPC_Cap-C_Gata6; Mus musculus; OTHER
library_construction_protocol: Capture-C data was processed and analyzed according to PMCID: PMC7613269. Briefly, 5 × 106 cells were crosslinked with 2% paraformaldehyde for 10 min. Subsequently, cells were quenched with 0.125 M glycine for 10 min. After washing three times with PBS, cells were resuspended in lysis buffer (10 mM Tris pH 8, 10 mM NaCl, 0.2% NP-40 and 1× protease inhibitors) for 30 min on ice. Afterwards, nuclei were pelleted by centrifugation and resuspended in NEB Buffer DpnII (B0543S, NEB) for subsequent chromatin digestion with DpnII (R0543S, NEB) followed by proximity ligation (Life Tech, cat. no. EL0013). Next, de-crosslinking was performed with Proteinase K (3U; Thermo Fisher, cat. no. EO0491) and subsequent incubation with RNase A (7.5 mU; Roche: 1119915). DNA was purified using the Qiagen kit (28506). Purified DNA was sonicated using Covaris S220 Focused-ultrasonicator (time, 180 s; duty factor, 10%; peak incident power, 175 W; cycles per burst, 200) to an average size of 200 bp. Sheared DNA was purified using AMPure XP SPRI beads (Beckman Coulter, cat. no. A63881). 3C libraries were generated using the NEBNext Ultra II kit (New England Biolabs, cat. no. E7645S/L). Index primers set 1 and 2 from the NEBNext Multiplex Oligos for Illumina kit (New England Biolabs, E7335S/L E7500S/L) were incorporated using Herculase II Fusion Polymerase Kit (Agilent, cat. no. 600677). Enrichment of ligated fragments containing the viewpoints of interest was performed using 120bp long probes in two consecutive rounds, as described for MCC in PMID: 34108683. Isolation of hybridized DNA was performed using M-270 Streptavidin Dynabeads (Invitrogen, 65305) and amplified by PCR (11 cycles). The second enrichment reaction was performed using the pulled down material from the first enrichment reaction as input. The quality of the final libraries was assessed with a fragment analyzer and sequenced using the the Novaseq6000 sequencing system (150bp paired-end reads;15 Mreads/lib).