GSM9096810: Wdr4^cKO.input; Mus musculus; RNA-Seq
For input RNA, total RNA was extracted using TRIzol reagent (Invitrogen) according to the manufacturer's protocol. Briefly, cells were lysed in TRIzol, followed by the addition of chloroform and centrifugation to separate the aqueous and organic phases. Under acidic conditions, RNA partitions into the aqueous phase, which was collected and precipitated with isopropanol to recover RNA.  For footprint RNA, ribosome profiling was performed based on a standard protocol with minor modifications. Cell lysates were mechanically sheared, clarified by centrifugation, and treated with RNase I and DNase I to digest unprotected RNA and DNA. Digestion was stopped with SUPERase·In, and ribosome-protected fragments (RFs) were purified using MicroSpin S-400 HR columns and size-selected (>17 nt) with the RNA Clean & Concentrator-25 kit (Zymo Research). rRNA was depleted by hybridization with antisense DNA probes followed by RNase H and DNase I digestion. Final RFs were purified using magnetic beads (Vazyme). For input RNA, mRNA was enriched using mRNA Capture Beads, followed by heat-induced fragmentation. First-strand cDNA synthesis was performed using a reverse transcription enzyme mix, and during second-strand synthesis, end repair and A-tailing were completed. Adaptors were then ligated, and target fragments were selected using Hieff NGS® DNA Selection Beads. PCR amplification was performed to generate the final libraries, which were sequenced on the Illumina NovaSeq X Plus platform.  For footprint RNA, libraries were constructed using the NEBNext® Multiplex Small RNA Library Prep Set for Illumina® (E7300S/L). Adapters were ligated to both ends of ribosome-protected fragments (RFs), followed by reverse transcription and PCR amplification. PCR products of 140–160 bp were size-selected and used to generate cDNA libraries, which were sequenced on the Illumina NovaSeq X Plus platform by Gene Denovo Biotechnology Co. (Guangzhou, China).