GSM8910926: AA(-)_2; Homo sapiens; RNA-Seq
A sucrose density gradient of 15-45% (w/v) was newly made in a SW41 ultracentrifuge tube from Beckman (Indianapolis, IN). Cells were broken down in a solution containing polysome lysis buffer (100 mmol·L-1 KCl, 100 mg·ml-1 cycloheximide, 2% Triton X-100, 5 mmol·L-1 MgCl2, 10 mmol·L-1 HEPES, pH 7.4), and then placed on sucrose gradients for centrifugation at 36,000 × g for 2 hours. A portion of each ribosome fraction was used to isolate total RNA using TRIzol™ reagent. Illumina TruSeq RNA Sample Prep Kit (Cat#FC-122-1001) was used with 1 μg of total RNA for the construction of sequencing libraries. RNA libraries were prepared for sequencing using standard Illumina protocols.