GSM9476471: HEK293T, FLAG-DHX29, rep1; Homo sapiens; OTHER
Cells were washed with cold-PBS, UV-crosslinked (254 nm, 300 mJ/cm²), and harvested in lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 10 mM DTT, 5 mM MgCl₂, 1% TritonX-100). Lysates were DNase-treated (TurboDNase, 10 min, on ice), clarified (20,000×g, 10 min, 4°C), and digested with RNase I (0.01 units/ml, 37°C, 5 min). After SDS addition (0.1%), lysates were immunoprecipitated with FLAG M2-Protein G Dynabeads (2h, 4°C). Beads were washed, and RNA-protein complexes were dephosphorylated (T4 PNK, 1h, 37°C) and ligated to IR800-conjugated 3′ adapters.  Complexes were eluted, resolved on SuperSep Ace 5-20% gel, and fragments >130 kDa were excised. RNA was extracted by Proteinase K digestion and isopropanol precipitation. Reverse transcription was performed using SuperScript IV (50°C, 60 min). cDNA fragments (10-200 nt) were size-selected, ligated to 5′ adapter (5′-/5Phos/NNAGATCGAAGGCAGCGCTGTGGAGAAAG/3ddC/-3′), and PCR-amplified (18 cycles, Phusion polymerase). Libraries were sequenced on Illumina NovaSeq X Plus.