GSM4455588: 4C_DMPK_d11_GM02180_rep1; Homo sapiens; OTHER
Samples of 10 million cells were crosslinked in 2% formaldehyde for 10 minutes at room temperature and quenched with glycine to a final concentration 0.13 M. Cross-linked samples were rinsed once with PBS and were either used immediately or flash-frozen in liquid nitrogen and stored at -80ºC for later use. Cells were lysed for 15 minutes on ice in lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.5% NP-40, 1% Triton X-100, 1x protease inhibitors). The first digestion was performed with 200 U DpnII (NEB) and incubating for 4 hours at 37ºC, then 200 U DpnII and overnight incubation at 37ºC, and finally 200 U DpnII and incubating 4 hours at 37ºC. DpnII digestion efficiency was assessed by agarose gel electrophoresis and DpnII was subsequently heat inactivated. The first ligation was performed at 16ºC overnight with 50 U T4 DNA ligase (Thermo Scientific) in 7 mL. Ligated samples were de-crosslinked with 30 µL Proteinase K (10 mg/mL) at 65ºC overnight followed by RNAse A treatment and phenol-chloroform purification. The second digestion was performed with 50 U of BfaI (NEB) at 37ºC overnight followed by BfaI heat inactivation. The second ligation was performed at 16ºC overnight with 100 U T4 DNA ligase in 14 mL. The resulting 4C template samples were precipitated and purified with the QIAquick PCR purification kit. 4C libraries were generated by amplifying 1 µg total of purified 4C template using the Expand Long Template PCR System (Roche) with 4C viewpoint primers with Illumina sequencing adapters that permitted multiplexing (in 50 µL PCRs), pooling reactions and purifying the PCR products with AMPure XP beads to exclude products less than 130 bp. Single-end sequencing of pooled 4C libraries was performed on Illumina HiSeq 2500.
GEO Accession
GSM4455588