GSM8633910: HeLa DDX39A-Tet-On cells, without HU treatment, without replicate; Homo sapiens; OTHER
The cells were harvested by scraping, washed twice with prechilled PBS and then fixed with 90% methanol at 4°C overnight. The cells were then washed twice with PBS, permeabilized with PBS containing 0.25% Triton-X 100 on a rotator at room temperature for 30 minutes. After centrifugation, the cell pellets were washed once with PBS. Then, fresh click reaction mixture (PBS with sequential addition and mixture of 25 μM biotin-PEG3-azide, 100 mM sodium ascorbate, 100 mM CuSO4) was added to the pellets, which were incubated on a rotator at room temperature for 2 hours. The cell pellets were then washed once with PBS and lysed on ice in lysis buffer (double-distilled water (ddH2O) containing 1% SDS, 50 mM Tris-HCl (pH 8.0)) for 10 minutes. A noncontact ultrasonic disruptor (Covaris M220 focused ultrasonicator) was subsequently used to sonicate the pellets for 3 minutes to obtain smeared DNA fragments of 100-300 bp. After sonication, the samples were centrifuged at 15000 rpm at 4°C for 10 minutes, after which the supernatant was collected. Part of the supernatant was taken as the input sample, while the remaining portion was used as the immunoprecipitation (IP) fraction. 100 μL of streptavidin beads was added to the supernatant of each IP group, and the mixture was incubated on a rotator at room temperature for 2 hours. The beads were subsequently washed successively with the following solutions: lysis buffer, 1 M NaCl, lysis buffer, 1 M NaCl, lysis buffer, and TE buffer (1 mM EDTA, pH 8.0; and 10 mM Tris-HCl, pH 8.0). Then, 300 μL of 2% β-mercaptoethanol was added to the beads, and the mixture was incubated with rotation at room temperature for 1 hour. The samples were subsequently centrifuged at 15000 rpm for 2 minutes, the supernatants were collected and extracted with phenol-chloroform. Then, 10 μL of glycogen (20 mg/mL), 100 μL of ammonium acetate (7.5 M), 40 μL of NaCl (5 M), and a 5x volume of ethanol were added to each supernatant and mixed, and the mixtures were incubated at -20°C overnight. The next day, the samples were centrifuged at 15000 rpm for 30 minutes at 4°C. We washed each pellet once with 75% ethanol and then centrifuged the sample and removed the supernatant. Finally, we resuspended the pellet in nuclease-free water to prepare the sample for sequencing. The purified DNA was used NEBNext® Ultra™ II DNA Library Prep Kit (NEB, E7645L) for library preparation. As for strand-specific groups, the DNA libraries were building with the Hieff NGS® Ultima Dual-mode RNA Library Prep Kit (Yeasen), DNA samples are started directly from the second cDNA synthesis step, skipping the first cDNA synthesis step.