GSM7061491: K562 cells, SF3B1 wild type, 4 days, replicate 2 [TTTLseq_K562_WT_Replicate2]; Homo sapiens; OTHER
Cells were harvested and washed in ice cold PBS and RNA extracted by Trizol and chloroform extraction, ethanol precipitation. Total RNA was resuspended and treated with DNase, then extracted with acidic phenol:chloroform:isoamyl alcohol and again ethanol precipitated. RNA was resuspended in water and concentration was determined. Up to 150 mg of RNA was transferred to a new tube and 5% (w/w) of Drosophila S2 spike-in RNA was added. RNA was then chemically fragmented by mixing with 2x RNA fragmentation buffer. Fragmented RNA was incubated in Biotinylation Solution (20 mM Tris pH 7.5, 2 mM EDTA pH 8.0, 40% dimethylformamide, 200 mg/ml EZ-link HPDP Biotin (Thermo Scientific, 21341)) for 2h in the dark at 25°C, 800 rpm. After chloroform: isoamyl alcohol (24:1) extraction followed by purification (Qiagen RNeasy Mini Kit, 74104), the biotinylated RNA was resuspended in water and biotinylated-RNA was isolated using Dynabeads MyOne Streptavidin C1 magnetic beads. s4U-biotinylated RNA was eluted from the beads by cleaving the HPDP Biotin in elution buffer and cleaned up using 1x RNAcleanXP beads. For time lapse chemistry, isolated s4U-enriched RNA was added to a mixture of 3M sodium acetate pH 5.2, EDTA, RNAse free water, and trifluoroethylamine. NaIO4 was then added dropwise, and the reaction mixture was incubated for 1 h at 45 °C. 1x volume of RNA clean beads were then added and purified. 2 μl of reducing master mix was added to each sample for a final volume of 20 μl, mixed, and incubated in thermocycler at 37C for 30 min. The mixture was then mixed with 1x RNAclean beads and RNA purified Eluted RNA was quantified with Qubit High Sensitivity Assay kit and used to produce barcoded cDNA sequencing libraries using the SMARTer® Universal Low Input RNA Kit