title: GSM9221460: Input_2; Homo sapiens; RIP-Seq
library_construction_protocol: For T&T-seq transcriptome RNA extraction, 100,000 oocyte-like cells (OLCs) were lysed in 2 μL aliquot, mixed with 1 mL TRIzol Reagent (Invitrogen #15596026), vortexed, incubated 5 min at RT, followed by chloroform addition (200 μL), centrifugation (12,000 × g, 15 min, 4°C), isopropanol precipitation (500 μL, 10 min RT), ethanol wash (75%), and resuspension in 11 μL nuclease-free water for storage at -80°C. For T&T-seq translatome RNA extraction, 8 μL lysate from 100,000 OLCs underwent ribosome capture using RiboLace Beads (Immagina) with 70-min rotation at 4°C, washed twice with W-Buffer, dissociated in 1% SDS + Proteinase K (37°C, 30 min), then extracted identically to transcriptome RNA including TRIzol/chloroform separation and overnight ethanol precipitation before resuspension. For RIP-seq RNA extraction, 1,000,000 OLCs were lysed in RIP buffer (50 mM Tris-HCl pH 7.4, 100 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate) with protease/RNase inhibitors, precleared with Protein G Dynabeads (Thermo #10004D), immunoprecipitated with 5 μg BOLL antibody (Proteintech #13720-1-AP) overnight at 4°C followed by 4-h bead binding, washed four times with lysis buffer, resuspended in 1 mL TRIzol for RNA isolation via chloroform/isopropanol method, and eluted in 20 μL nuclease-free water. All RNA extracts were stored at -80°C pending library construction. RNA libraries for T&T-seq and RIP-seq were prepared using SMART-Seq2 library construction strategy.