GSM9271116: WTC11iCRISPRa_4C_sgDLX1_DLX1pe_rep2; Homo sapiens; OTHER
10 million cells were crosslinked for 10 minutes with 2% formaldehyde. Crosslinking was quenched with 0.13 M glycine, and nuclei were isolated in lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5 % IGEPAL CA-630, 1 % TritonX-100, 5 mM EDTA, and protease inhibitors) for 20 min. Chromatin was digested overnight with 200 U DpnII (NEB), the primary restriction enzyme, then overnight ligated with 50 U of DNA ligase (Invitrogen) following published protocols (Krijger et al. 2020; Taylor et al. 2022; Ray-Jones et al. 2025). Crosslink were reversed with proteinase K, and 3C templates were purified with AMPure XP beads (Beckman Coulter). Up to 60 μg of template was diluted to 100 ng/μL, and digested overnight with NlaIII (NEB, 1 U/μg DNA template), the secondary restriction enzyme, followed by a second overnight ligation at a final DNA concentration of 5 ng/μL using 2 U/μg T4 DNA ligase (Invitrogen). DNA was purified by Phenol Chloroform Isoamyl Alcohol (P:C:I) extraction and AMPure XP beads, according to (Ray-Jones et al. 2025). 4C libraries were generated in two rounds of PCR, as described in (Krijger et al. 2020). Briefly, first-round PCRs (16 cycles) were performed with Expand Long Template polymerase (Roche) on four 200-ng reactions per sample, followed by pooling and 0.8 X AMPure XP beads cleanup to remove long primers. A second PCR (12 cycles) was performed with up to 100 ng of template per reaction to introduce Illumina sequencing adapters. Finally, libraries were purified as above and sequenced as 60-bp paired-end reads.