title: GSM8804959: PR_TT_rep2; Homo sapiens; OTHER
library_construction_protocol: After rinsing twice with ice cold PBS, 1 mL TRIzol reagent (Thermo Fisher Scientific) was added to lyse cells. After incubation at room temperature for 5 min, 200 uL chloroform was added. Samples were vortexed and incubated briefly at room temperature and centrifuged at 12,000 × g, 5 min, 4°C. The aqueous phase was transferred to a new tube and total RNA was isolated by isopropanol precipitation. Genomic DNA was removed using the TURBO DNase Kit (Ambion) and then samples was further cleaned up with RNAclean XP beads (Beckman) following the manufacturer's instructions. 75 ug purified RNA was sheared in 160 uL RNA fragmentation buffer (75 mM Tris pH 7.4, 112.5 mM KCl, 4.5 mM MgCl2) for 3.5 min at 94°C. Sheared RNA was purified with the RNeasy Mini Kit (Qiagen). For each reaction, 50 ug purified RNA was biotinylated by 5 ug MTSEA-biotin-XX (Biotium) for 30 min at room temperature with rotation. Samples were purified again with RNeasy Mini Kit (Qiagen) to remove unreacted MTSEA-biotin-XX. Purified biotinylated RNA was incubated with 10 uL blocked Dynabeads MyOne Streptavidin C1 beads (Thermo Fisher Scientific) for 15 min at room temperature with rotation. Beads were washed with 100 uL High-salt Wash Buffer (100 mM Tris pH 7.4, 10 mM EDTA, 1M NaCl, 0.05% Tween-20) three times and 100 uL TE Buffer (10 mM Tris pH 7.4, 1 mM EDTA) twice. Nascent transcript was eluted in 25 uL Elution Buffer (100 mM DTT, 20 mM HEPES pH 7.4, 1mM EDTA, 100 mM NaCl, 0.05% Tween-20) for 15 min at room temperature with rotation. 20 uL Eluted RNA was added with 0.835 uL 3M sodium acetate pH 5.2, 0.2 uL 500 mM DETA, 1.3 uL TFEA, 1.365 uL waster, 1.5 uL 200mM mCPBA for TimeLapse chemistry, and then purified with 25 uL RNAclean XP beads (Beckman). 18 uL Purified RNA was added with 0.8 uL water, 0.2 uL 1 M DTT, 0.2 uL 1M Tris pH 7.4, 0.4 uL 0.5 M EDTA, 0.4 uL 5 M NaCl for reducing treatment, and then purified with 20 uL RNAclean XP beads (Beckman). Purified RNA was used for sequencing library prep with the KAPA RNA HyperPrep Kit with RiboErase (Roche KR1351).
identifiers.namespace: GEO
identifiers.id: GSM8804959_r1