GSM8968845: EV, +auxin, Rep 3; Homo sapiens; RNA-Seq
RNA was extracted using TRIzol (MRC Cat#TR 118). RNA was then treated with RQ1 DNase, and then rRNA was removed using the RiboMinus Eukaryote System V2. GLORI-Seq was performed as described in Shen et al Nature Protocols, 2024. In brief, RNA was fragmented at 94°C for 4 min using the NEBNext Magnesium RNA Fragmentation Module to ~100-200 nt. Then, guanosines were protected, non-methylated adenosines were deaminated to inosines, and guanosines were deprotected. End repair was performed using Antarctic phosphatase and T4 PNK. Then, libraries were made using the NEBNext Small RNA Library Prep Set for Illumina with the NEBNext Multiplex Oligos for Illumina, Index primers set 1 and 2. Libraries were purified using the Monarch Spin PCR & DNA Cleanup Kit and a 1.5X KAPA Pure Beads cleanup. Finally, 150x150 paired-end sequencing was done on a NovaSeq X 1.5B flow cell with 10% PhiX added to diversify. Note 3 biological replicates were performed for each experiment.