4C of Undifferentiated LUHMES 3 PWAR1
We used 3 technical replicates for each viewpoint (VP) and condition harvested from a separate LUHMES cell thaw passage 4 with 10 million cells each.  The 4C protocol was adapted from Krijger et al (2020) with the following modifications: Invitrogen MagMax DNAbinding beads (Cat. No. 4489112) were substituted for the Nucleomag beads (42).  Primary restriction enzyme digest was performed using DpnII (Cat. No. R0543S) and secondary digestion with CviQI (Cat. No. R0639S) from NEB.  Before sequencing a final cleanup using SPRIselect beads from Beckman Coulter (Cat No. B23317) was used.  The fragment size distribution of the library pool was verified via micro-capillary gel electrophoresis on a Bioanalyzer 2100 (Agilent, Santa Clara, CA). The pool was bead cleaned twice to remove the adapter-dimer at 129 bp. Then, the library was quantified by qPCR with a Kapa Library Quant kit (Kapa Biosystems/Roche, Basel, Switzerland). The library was sequenced on one flow cell of Aviti sequencer (Element Biosciences, San Diego, CA) with single-end 150 bp reads. Pipe4C was used for the initial bioinformatic analysis followed by peak calling with PeakC using the default settings aligned to hg19 (4244).  Viewpoint (VP) primers included: SNRPN VP reading primer (FP) 5-TGTAATCCCAACACACTGG-3 and non-reading primer (RP) 5-TGTTGTCTCTCATTTTCCTCA-3.  For the PWAR1 VP FP 5-TCATAGCTGAAACCATGAGA-3 and RP 5-TAGACGAACATTGCTGTGAC-3 were used.  For the UBE3A viewpoint FP 5-ACCATCTTGGGAGACACAC-3 and RP 5-TCCTCATCTTGGTGGTAAAG-3 were utilized.