GSM9097009: GNL3L-His-FLAG, CRAC - Protein of interest, Replicate 2; Homo sapiens; OTHER
HEK293 Flp-In cells expressing GNL3L-His₆-2xFLAG or the His₆-2xFLAG tag control were UV crosslinked (254 nm, 800 mJ/cm²), lysed by sonication in TMN150 buffer, and subjected to anti-FLAG and Ni-NTA affinity purification. After SDS-PAGE and transfer to nitrocellulose, labeled protein–RNA complexes were visualized by autoradiography. Membrane regions containing crosslinked material were excised and treated with Proteinase K (Roche). RNA was extracted using TRI-Reagent LS (Sigma-Aldrich) according to the manufacturer's instructions. Crosslinked protein–RNA complexes were partially digested with RNace-IT and purified via anti-FLAG and Ni-NTA affinity. RNA fragments were dephosphorylated with FastAP (Thermo Fisher) and ligated to a pre-adenylated Illumina RA3 3′ adapter (5′-/5rApp/TGGAATTCTCGGGTGCCAAGG/3ddC/-3′) using RNA ligase II (truncated KQ mutant, NEB). 5′ ends were labeled with [γ-32P] ATP and ligated to a 5′ adapter containing a 5-nt Unique Molecular Identifier (UMI) (5′-/InvddT/ACACrGrUrUrCrArGrArGrUrUrCrUrArCrArGUrCrCrGrArCrGrArUrCrNrNrNrNrNrArGrC-3′) using T4 ssRNA Ligase I (NEB). RNA  extracted from excised nitrocellulose membrane regions was reverse transcribed with SuperScript III (Thermo Fisher), PCR-amplified with Phusion Hot Start II DNA Polymerase and gel purified. Libraries were sequenced on an Illumina NovaSeq 6000.