title: GSM7094270: TTseq-HCT116-LEO1-607-P-A; Homo sapiens; OTHER
library_construction_protocol: Following HaloPROTAC3 treatment, cells were collected and lysed with Trizol reagent. Total RNA was extracted according to the manufacturer's instructions. TT-seq was performed as previously described (Gregersen et al., 2020; Schwalb et al., 2016) with some modifications. Cells were labeled in media with 400 μM 4-thiouridine (4sU) (Sigma-Aldrich, Cat# T4509) for 10 min before harvest, and total RNA was extracted with TRIzol (Invitrogen, Cat# 15596-018), the labeled RNA (100 μg) was fragmented with NaOH and spike-in with 10% S2 RNA (10 μg). After biotinylated in a 300 μL reaction (100 μg RNA, 10mM HEPES-KOH, pH 7.5, 1mM EDTA, 0.167mg/ml MTSEA-biotin-XX (Sigma-Aldrich, Cat# M9938)), enriched the biotin-labeled RNA with Streptavidin M280 beads (Invitrogen, Cat# 11206D) and eluted by 100mM DTT. RNA was purified by 1.8 x RNAClean XP beads before library preparation.