title: GSM5963947: SHEP MYCN-ER FP-TT-Seq KL-2 rep3; Homo sapiens; OTHER
library_construction_protocol: For TT-seq, total RNA extraction with Trizol reagent. For TT-seq, the extracted total RNA was spiked-in with 4sU-labeled Drosophila S2 RNA. RNA was fragmented by 0.2 M NaOH, neutralized by 1× volume of 1 M Tris-HCl (pH 6.8) and precipitated with isopropyl alcohol. 100 μg of total RNA was subjected to biotinylation reaction in a total volume of 250 μl containing 10 mM HEPES (pH 7.5), 1 mM EDTA, and 5 ug of biotin-XX-MTSEA at room temperature for 30 minutes. The RNA was purified with Sera Mag carboxylate modified magnetic beads. The biotin-labeled RNA was further purified with the MyOne streptavidin C1 dynabeads. 4sU-RNA was eluted with DTT and subjected to strand-specific RNA-Seq library preparation prior to next generation sequencing on a NovaSeq 6000.