GSM8241170: HepG2 WDR4 PARCLIP rep2; Homo sapiens; OTHER
3 volumes of the lysis buffer (50 mM HEPES, pH 7.5; 150 mM KCl; 2 mM EDTA; 0.5% (v/v) NP-40, with 1:100 protease inhibitor (Roche) and 40 U ml-1 RNasin® Ribonuclease Inhibitors (Promega) added freshly) was added to the cell pellet and incubated on ice for 10 min with periodic perturbation. The cell lysate was then centrifuged at 15,000 g for 15 min and the clear supernatant was collected. RNase T1 (1,000 U µL-1, Thermo Fisher Scientific) was added to the clear lysate to a final concentration of 0.1 U µL-1 and an incubation at room temperature was performed for 15 min. The reaction was then quenched on ice. After 5 min, antibodies were added to each sample (5 µg or according to the manufacturer's protocols). The antibody and the lysate were incubated at 4 °C for 2 hours under periodic rotation. Protein A beads or protein G beads (Thermo Fisher Scientific) were washed (50 µL for each sample, or adjusted to the antibodies amount) with IP wash buffer (50 mM HEPES, pH 7.5, 300 mM KCl, 0.05% (v/v) NP-40, with 1:100 protease inhibitor (Roche) and 40 U ml-1 RNasin® Ribonuclease Inhibitors (Promega) added freshly) for 2 times. The beads were resuspended in 50 µL lysis buffer for each sample and added to the antibody-lysate mixture subsequently. Another two-hour incubation at 4 °C was performed with low-speed rotation. After the incubation, the beads were washed three times with IP wash buffer (50 mM HEPES, pH 7.5, 300 mM KCl, 0.05% (v/v) NP-40, with 1:100 protease inhibitor (Roche) added freshly) and then resuspended with 200 µL IP wash buffer per sample. The beads were treated with a second round of RNase T1 digestion under a final concentration of 10 U µL-1 for 15 min at room temperature. The reaction was then quenched with the addition of 10 µL SUPERase-In followed by a 5-min incubation on ice. The beads were washed three times with high-salt wash buffer (50 mM HEPES, pH 7.5, 500 mM KCl, 0.05% (v/v) NP-40, with 1:100 protease inhibitor (Roche) added freshly) and twice with 1X PNK buffer (NEB) afterward. The beads were resuspended with 200 µL of 1X PNK buffer (NEB) and underwent T4 PNK (Thermo Fisher Scientific) end repair with standard procedures as previously mentioned under 37 °C. After the incubation, the beads were washed once with 1X PNK buffer followed by proteinase K digestion as described before. The RNA was recovered with RNA Clean & Concentrator (Zymo Research) before library construction. Libraries were constructed followed the commerical protocol for NEBNext Small RNA Library Prep Set for Illumina (NEB). All libraries were sequenced on Illumina Nova Seq with single-end 100 bp read length.