GSM6956516: 4Cseq-hg-Control-FB-LBX1-Rep2-L21108; Homo sapiens; OTHER
Skin biopsies were collected from SHFM3 patients and controls by standard procedures. Fibroblasts were cultured in DMEM (Lonza) supplemented with 10% fetal calf serum (Gibco), 1% L-glutamine (Lonza) and 1% penicillin/streptomycin (Lonza). For 4C-seq libraries fixation and lysis were performed as described for Capture Hi-C. After the first digestion with DpnII (NEB), sticky ends were religated in a 50 ml falcon tube (700 μl 10 ligation buffer (Fermentas), 7 ml H2O, 50 U T4 DNA ligase (Thermo); overnight at 16 °C) and DNA de-cross linked and cleaned as described for the Capture Hi-C. Next, a second digestion (150 μl sample, 50 μl 10× Csp6I buffer (Thermo), 60 U Csp6I (Thermo) 295 μl H2O; overnight at 37 °C) and another re-ligation were performed. For all viewpoints, DNA was purified using a PCR clean up Kit (Qiagen) and 1.6 μg DNA was amplified by PCR (LBX1 Viewpoint: read-primer 5′-TCTATATGCTACCATGATC-3′, secondary-primer 5′-GATGAACTGGAATACCCA-3′; FGF8 Viewpoint: read-primer 5′-AGGGTGCGTTCCAAGATC-3′, secondary-primer 5′-GGTGGCCTGGATGGAAGT-3′; BTRC Viewpoint: read-primer 5′-CAACGCAGCGCCCGGATC-3′, secondary-primer 5′-CTGGGAATGAGGACCTAGGGC-3′). For the library reaction, primers were modified with TruSeq adapters (Illumina): Adapter1 5′-CTACACGACGCTCTTCCGATCT-3′ and Adapter2 5′-CAGAC GTGTGCTCTTCCGATCT-3′. Between 50 and 200 ng were used as input of a single 4C PCR reaction depending on the complexity. The reaction was performed in a 50 μl volume using the Expand Long Template System (Roche) and 29 reaction cycles. After the PCR all reactions were combined and the DNA purified with a PCR clean up Kit (Qiagen).