title: GSM8799074: KO_input_1; Homo sapiens; RIP-Seq
library_construction_protocol: Briefly, the protein G Dynabeads (Thermo Fisher Scientific) were washed and resuspended in the dsRIP lysis buffer (50 mM Tris-Cl, pH 7.4, 100 mM NaCl, 3 mM MgCl2, 0.5% IGEPAL CA-630). For each sample, 25 μL of beads were pre-bound to 5 μg of anti-dsRNA mAb (J2) by incubation for 2 h at 4 ℃. HeLa WT and KO cells were collected and washed with ice cold PBS for three times. Then, 3×10^7 cells for each group were lysed in 0.6 mL dsRIP lysis buffer for exactly 5 min on ice. Total RNA was harvested from the rest of cells using Trizol for both groups to serve as input samples and stored at -80 ℃. After that, the cell lysates were spin down by centrifugation at 13 000 g for 10 min at 4  ℃. For immunoprecipitation, 25 μL of J2-bound Protein G Dynabeads were added to the lysate and incubated for 1 h at 4 ℃ with rotation. Then, the beads were washed four times with dsRIP lysis buffer, and J2-bound dsRNA was harvested directly from beads with Trizol with a ratio of 1:5 of chloroform added. Briefly, both the input and dsRIP samples were used for library generation. Sequencing libraries were prepared using the Ribo-clean rRNA Depletion Kit (Vazyme, China, Cat# RN416) and KCTM Digital Stranded mRNA Library Prep Kit (Wuhan Seqhealth Co., Ltd. China) following the manufacturer's instruction. Subsequently, the libraries were quantified by Qubit 3.0 and sequenced on a DNBSEQ-T7 platform (MGI) using the PE150 sequencing mode to generate paired-end reads.