GSM7855162: Torin1-treated sample, 1h, Fraction 5; Homo sapiens; RNA-Seq
1h after treatment, cells were spun at 1000 x g for 1 min at room temperature, the media aspirated, and the cell pellet directly lysed in 150 µL of sucrose gradient lysis buffer. Lysates were gently pipetted to homogenize, transferred to ice for 10 min, and clarified by centrifuging at 8000 x g at 4°C for 5 min. 120 µg of RNA was sedimented along 10-50% sucrose gradients for 75 min and subsequently fractionated to 8 fractions. Thirty pg of eGFP mRNA was spiked into each fraction and RNA was extracted using TRIzol according to the manufacturer's instructions. Five µg of extracted RNA from each fraction was subjected to polyA-enrichment using the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB E7490) according to the manufacturer's instructions. One ng of polyA-enriched RNA from each sample was subjected to the Nanopore PCR-cDNA Barcoding kit (ONT SQK-PCB109) according to the manufacturer's instructions.