GSM5057848: Vector-polysome-seq_2; Homo sapiens; RNA-Seq
Cells transfected with different plasmids  were pretreated with 100 mg/ml cycloheximide for 30 min at 37°C prior to washing with ice-cold PBS containing 100 mg/ml cycloheximide. Cells were then lysed in polysome lysis buffer (20 mM Tris-HCl (pH 7.4), 100 mM KCl, 5 mM MgCl2, 100 mg/ml cycloheximide and 1% NP40). After 20 min on ice, the lysates were centrifuged (12,000 rpm for 15 min at 4°C), and the supernatants were loaded onto 10-50% sucrose gradients and centrifuged for 15 h at 25,000 rpm and 4°C in a SW41 rotor. Separated samples were fractionated at 0.75 ml/min through an automated fractionation system that continually monitors OD254 values. An aliquot of the ribosomal fraction was used for extraction of total RNA with TRIzol reagent (Yeasen). Purified RNAs were used for cDNA synthesis using the Smart-seq2 method (Picelli et al., 2014). Briefly, the polyadenylated mRNAs were captured usingby the PolyT primers. After 3 min lysis at 72°C, the Smart-seq2 reverse transcription reactions were performed. After the first-strand reaction, the cDNA was amplified using a limited number of cycles (12∼14 cycles). Sequencing libraries were constructed from 1 ng of amplified cDNA using the TruePrep DNA Library Prep Kit V2 for Illumina (Vazyme, TD503) according to the manufacturer's instructions. Barcoded libraries were pooled and sequenced on the Illumina HiSeq X Ten platform in the 150 bp paired-end mode.