title: GSM1973200: HCT116 bait 5 low cell input, rep6; Drosophila melanogaster; Homo sapiens; OTHER
library_construction_protocol: Chromosome Conformation Capture (3C) was performed as described before with slight modification. Briefly, 1 million of the cells were fixed with 1% monomeric formaldehyde for 10 min at RT and excess amount of the formaldehyde was captured by 0.125 M Glycine. Then, cells were collected and suspended in 10 ml of nuclear isolation buffer (0.2% NP-40, 10 mM Tris-cl, pH 7.5, 10 mM NaCl, x1 Proteinase inhibitor (Roche, 04693116001)) and incubated for 10 min on ice. Isolated nuclei were precipitated and washed with 1 ml of Buffer 2 (New England Biolabs, B7002S). Next, nuclei were re-suspended in 500 ul of x1.2 buffer 2. Un-crosslinked fraction was removed by adding 5 ul of 10% SDS and incubated for 1 hr at 37 °C. Then, excess amount of SDS was captured by TritonX-100. The chromatin DNA was fragmented by the incubation at 37 °C for overnight with HindIII (1 U/ul, New England Biolabs, R0104T). As a spiking control, same amount of chromatin was isolated from Drosophila S2 cells with same method and mixed with chromatin for analysis. After the inactivation of the enzyme, the solution was transferred to 50 ml tube containing 7 ml of ligation buffer (50 mM Tris-cl, pH 7.5, 10 mM MgCl2, 1% Triton X-100) and incubated for 1 hr at 37 °C. Then, 70 ul of 100 mM ATP, 70 ul of 1 M DTT and 2000 U of T4 DNA ligase were added. Then, the solution was incubated at 16 °C for overnight followed by for 1 hr at RT. Crosslink was reversed with conventional methods. Finally, DNA was collected by ethanol precipitation followed by the purification with MSB Spin PCRapace (Stratec, 1020220200) and Zymo Clean & Concentrator-5 (Zymo Research, D4013). The digestion efficiency was estimated by qPCR. The quality of 3C DNA was visualized on 1% agarose gel. 3C DNA was tagmented and adapter DNA which equipped with T7 RNA polymerase promoter was incorporated by limited cycle PCR using either Nextera or Nextera XT DNA sample prep kit (Illumina, FC-121-1031, FC-131-1024). The resulting product was amplified by in vitro transcription (Maxiscript T7 kit, Life Technology, AM1312M). The template DNA was fragmented by Turbo DNase I (Life Technology, AM2239) prior to the RNA purification by RNeasy (Qiagen, 74104). For primer annealing, 1 ug of template RNA with 0.2uM of bait-specific primer was denatured at 75 °C for 3 min and then mixed with x2 buffer of Platinum Quantitative RT-PCR ThermoScript One-Step System (Life Technology, 11731-015) and 0.5U/ul of SUPERase In RNase inhibitor (Life Technology, AM2694). The solution was incubated at 4 °C for 44-48 hrs. Then, following the incubation at 65 °C for 5 min, the primer was extended by adding Thermoscript Plus/Platinum Taq mix at 60 °C for 30 min. After the inactivation of reverse transcriptase, bait-interactor chimeric DNA was enriched by PCR with Illumina P5 and bait-specific primer. At last, Illumina P7 sequence was incorporated with Nextera DNA sample prep kit by following manufacturer's instruction.
attributes.tag: GEO Accession
attributes.value: GSM1973200