title: GSM4990438: a12_Hec1b_shNRSF; Homo sapiens; OTHER
library_construction_protocol: Cell pellets were lysed twice with cold lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 5 mM EDTA, 0.5% NP-40, 1xprotease inhibitors) for nuclei. Then, Nuclear pellet was resuspended in 225 μl of water, 30 μl of 10x DpnII buffer, and 7.5 μl of 10% SDS and incubated at 37 °C for 1 h with constant shaking at 900 rpm. After quenching SDS by adding 37.5 μl of 20% Triton X-100 and incubating at 37 °C for 1 h with shaking at 900 rpm. The nuclei were then digested with 10 μl of DpnII (10 U/μl) in situ at 37 °C with shaking at 900 rpm overnight. After enzyme inactivation at 65 °C for 20 min, nuclei were centrifuged at 2500g for 5 min, resuspended and digested in 358 μl of water, 40 μl of 10x T4 ligation buffer, and 2 μl of T4 DNA ligase (400 unit/μl) overnight at 16 °C. The ligated product was then reverse crosslinked by adding 10 μl of proteinase K (10 mg/ml) and heating at 65 °C for 4 h, followed by adding 2 μl of RNase A (37 mg/ml) and heating at 37 °C for 45 min. DNA was then extracted using phenol-chloroform. 2 μl of glycogen (20 mg/ml) was added to facilitate DNA precipitation. For 4C libraries, the precipitated DNA was dissolved in 50 μl water, and then sonicated using the Bioruptor system with low energy setting at a train of 30s on with 30s off for 12 cycles for DNA fragments ranging from 200 to 600 bp. With the fragmented DNA as template, a linearized amplification step was applied using a 5' biotin-tagged primer complementary to the viewpoint fragment in 100 μl of PCR system for a total of 80 cycles. The PCR product was denatured at 95 °C for 5 min and immediately chilled on ice to obtain ssDNA. The ssDNA was enriched and purified with Streptavidin Magnetic Beads (Invitrogen) according to the manufacturer's instructions, and then ligated with annealed adapters in 45 μl of ligation system (20 μl of DNA-coated beads, 4.5 μl of 10x T4 ligation buffer, 10 μl of 30% PEG 8000, 1 μl of 50 μΜ adapter, 0.9 μl of T4 DNA ligase, 8.6 μl of water). The beads were then washed twice to remove the free adapters with the B/W buffer (5mM Tris-HCl, 1M NaCl, 0.5mM EDTA, Ph7.5). The DNA-coated beads were resuspended in 10 μl water. With the DNA on beads as template and a pair of primers, the QHR-4C libraries were generated by PCR amplification and purified with a PCR purification kit (Roche).
attributes.tag: GEO Accession
attributes.value: GSM4990438