GSM7434393: Col-0, light, polysomal RNA, rep2; Arabidopsis thaliana; RNA-Seq
Polysomal RNA was isolated as describe in Liu et al., 20121. The 500 μL of ground sample powders were resuspended by 500 μL of polysome extraction buffer (200 mM Tris-HCl, pH 8.0, 50 mM KCl, 25 mM MgCl2, 50 µg/mL cycloheximide, 100 µg/mL heparin, 2% PTE, 10% DOC, 400 U/mL RNasin) and centrifuged at 12,000 xg at 4℃ for 10 minutes. The supernatants were filtered and loaded to 11 mL of sucrose gradient (12.5% to 60%) and then were ultracentrifuged at 210,000 xg at 4℃ for 3.5 h. The OD254 of gradients were detected by UV detector to illustrate the pattern of ribosomal RNA profiling. PL% is the percentage of the polysomal RNA relative to the sum NP and PL RNA. After the centrifugation, the NP and PL fractions were collected and extracted with PCI, precipitated by LiCl, and resuspended with Ultra-Pure water. The total RNAs were extracted by the pine tree method (Chang et al., 1993). TruSeq Stranded mRNA Library Prep Kit (Illumina, San Diego, CA, USA) was used to generate mRNA sequencing library according to the manufacturer's instructions.