GSM9382734: TT-chem Seq, 5h RNAPIIi, biol rep1; Homo sapiens; OTHER
Cells were washed with PBS and lysed in TRIzol. 1.2µg spike-in RNA was added per 10 million cells, resulting in addition of ~1% spike RNA per condition. RNA was isolated by TRIzol–chloroform isolation and ethanol precipitation. 400 μg RNA (in a total volume of 116 μl) was fragmented by adding 4 μl 5 M NaOH and incubating on ice for 30 min, then stopped by addition of 80 μl of 1 M Tris pH 7 and cleaned up twice using the Micro Bio-Spin P-30 Gel Columns (Bio-Rad, 7326223) according to the manufacturer's instructions. Biotinylation of 4-SU residues was performed in a total volume of 250 μl, containing 10 mM Tris–HCl pH 7.4, 1 mM EDTA and 5 µg of MTSEA biotin-XX linker (Biotium, BT90066) for 30 min at RT in the dark, followed by phenol–chloroform extraction and elution in 50ul water. 300 µg RNA (37.5 µl) was denatured by 10 min incubation at 65 °C and added to 100 μl μMACS Streptavidin MicroBeads (Milentyl, 130-074-101). RNA was incubated with beads for 15 min at RT and beads were applied to a μColumn in the magnetic field of a μMACS magnetic separator. The beads were washed three times with 55 °C pulldown wash buffer (100 mM Tris–HCl pH 7.4, 10 mM EDTA, 1 M NaCl and 0.1% Tween-20). Biotinylated RNA was eluted twice by addition of 100 mM DTT and cleaned up using the RNeasy MinElute kit (Qiagen, 74204) using 1,050 μl ethanol (≥99%) per 200 μl reaction after addition of 700 μl of RLT buffer to precipitate RNA of less than 200 nucleotides. 50 to 1300 ng of the purified 4SU-labelled RNA was then used as input for in-house stranded RNA library prep, similar to the NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina (NEB #E7760) without rRNA depletion. The library was amplified with ten PCR cycles and quality-control checked on the TapeStation (Agilent) using the High Sensitivity DNA Kit before pooling.