GSM8309813: U2OS_DRB_20minR_rep2_batch1; Homo sapiens; OTHER
Total RNA was harvested using TRIzol including an additional chloroform cleanup and isopropanol precipitated. 20 ug of total RNA were spiked in with 0.2 ug of yeast spike-ins (Saccharomyces cerevisiae W303a cells labeled with 5mM 4-thiouracil (4tU, Sigma, 440736) for 5 min, see Gregersen et al., 2022 for detailed procedure). RNA was fragmented for 20 min on ice by base hydrolysis and returned to a neutral pH buffer prior to biotinylation as previously described (Gregersen et al., 2020a). For addition of biotin to 4sU residues we used the MTSEA Biotin-XX linker (Biotium, BT90066). Biotinylated RNA was purified by phenol:chloroform and isopropanol precipitation, heat denatured and used for purification with uMACS Streptavidin microbeads (Miltenyi, 130-074-101). 4sU-RNA was eluted from beads using 2 rounds of 100 mM DTT and cleaned-up with the RNeasy MinElute Cleanup Kit (Qiagen, 74204) using 1.5x excess of ethanol for the RNA precipitation compared to the supplied protocol to ensure recovery of small RNA fragments. Concentration of 4sU-RNA was measured by Qubit (RNA high sensitivity assay, ThermoFisher Q32852) and the size range determined by high sensitivity RNA assay on a TapeStation (high sensitivity RNA ScreenTape, Agilent, 5067-5579). 4 ng of 4sU RNA was used for next-generation sequencing library preparation with the NEBNext Ultra II directional RNA kit (NEB, E7760) using Unique Dual Index UMI Adaptors (NEB, E7416) following the protocol for degraded RNA (RIN 1-2) without additional RNA fragmentation. Libraries were amplified using 12 PCR cycles, concentration measured by Qubit (DNA high sensitivity assay) and size distribution assessed using TapeStation (high sensitivity D5000 ScreenTape, Agilent, 5067-5592). The final library size distribution peaked at 390 nt, corresponding to an insert size of 260 nt. Libraries were sequenced on two P3 v100 flow cells (130 cycles) in paired-end mode on a NextSeq2000 (Illumina).