GSM9316592: BT549, TRMT112 OE Polysomes -2; Homo sapiens; RNA-Seq
Cells were washed twice with ice-cold PBS containing cycloheximide and lysed in polysome lysis buffer (20 mM Tris-HCl, 10 mM MgCl2, 300 mM NaCl, 1% Triton X-100, 100 µg/mL cycloheximide, 0.1 U/µL RNase inhibitor, 0.5 mg/mL heparin, and 1x Halt protease inhibitors). Lysates were clarified by centrifugation at 13,000 rpm for 10 minutes at 4°C. The protein concentration was measured, and 2.5 mg of total protein was loaded onto pre-prepared 10-50% sucrose gradients. Gradients were ultracentrifuged at 35,000 rpm for 3 hours 20 minutes at 4°C using a Beckman SW41Ti rotor. Following ultracentrifugation, gradients were fractionated using the Brandel SYN-202 Density Gradient Fractionation System. RNA was then isolated from the polysomes using the RNeasy Mini Kit, and 1 ug of total RNA was used for library construction and sequencing. The RNA sequencing libraries were prepared using the NEBNext Ultra II RNA Library Prep Kit for Illumina using manufacturer's instructions (New England Biolabs, Ipswich, MA, USA). Briefly, mRNAs were initially enriched with Oligod(T) beads. Enriched mRNAs were fragmented for 15 minutes at 94°C. First strand and second strand cDNA were subsequently synthesized. cDNA fragments were end repaired and adenylated at 3'ends, and universal adapters were ligated to cDNA fragments, followed by index addition and library enrichment by PCR with limited cycles. The sequencing library was validated on the Agilent TapeStation (Agilent Technologies, Palo Alto, CA, USA), and quantified by using Qubit 2.0 Fluorometer (ThermoFisher Scientific, Waltham, MA, USA) as well as by quantitative PCR (KAPA Biosystems, Wilmington, MA, USA).