title: GSM6940560: h32 nonpolysome ENDrep4; Arabidopsis thaliana; RNA-Seq
library_name: GSM6940560
library_construction_protocol: Approximately 0.25g of frozen seedling or inflorescence was ground into fine powder in liquid nitrogen. One milliliter of polysome extraction buffer (PEB) (0.2M Tris-HCl pH8.0, 0.2M KCl, 35mM MgCl2, 25mM EGTA, 1% TritonX-100, 2% polyoxyethylene-10-tridecyl ether (PTE), 100mM 2-Mercaptoethanol, 25ug/ml cycloheximide (CHX), 100ug/ml chloramphenicol (CHL) and 0.5mg/ml heparin) was added and mixed thoroughly with the powder. After 10 minutes incubation on ice, cell debris was removed by centrifugation for 5 min at 12,000 rpm in a microcentrifuge at 4 °C. Sodium deoxycholate (DOC) was added to the supernatant to a final concentration of 0.5%, and the lysate was kept on ice for 5 min. After centrifugation for 15 min at 12,000 rpm in a microcentrifuge, the supernatant was transferred to a new 1.5ml RNase-free tube and marked as PEB-input.  0.5 ml of the PEB-input was loaded onto a 4.4-ml sucrose gradient (15-55% Sucrose in 40mM Tris-HCl pH8.0, 20mM KCl, 10mM MgCl2, 25ug/ml CHX, 100ug/ml CHL, 0.5mg/ml heparin). After ultracentrifugation in a Beckman SW-55Ti rotor at 45,000 rpm for 65 minutes at 4°C, density gradient fractionation (Brandel, SYN-202) was performed as described before (94, 95). The fluid speed was 1.5 ml/min, and collection time for each fraction was 19 seconds. Ethylenediamine tetraacetic acid (EDTA) and sodium dodecyl sulfate (SDS) were added to 20mM and 0.5%, respectively, into each fraction at room temperature. RNA was immediately extracted from each fraction using TRI-reagent (MRC, TR118) according to the manufacturer's instructions. Pelleted RNA was resuspended in 25ul RNase-free ddH2O for library construction. The 18S-ENDseq library construction pipeline was adapted from the circular RT–PCR assay with modifications. 1.5ug of RNA was used for RNA self-ligation followed by reverse-transcription with the RT-primer 18c2, using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher, K1621). A custom-made universal primer and indexed primers were then employed to amplify 18S-END libraries, using the SYBR-Green qPCR Supermix (BioRad, 170-8882) in a CFX96 Real-Time system (BioRad, C1000 Touch) for 15 cycles. PCR products were size selected in an agarose gel and recovered by QIAEX II Gel Extraction Kit (150) (QIAGEN,20021), or directly purified with AMPure XP Beads (Beckman, A63881). After quality control and quantitation with a Bioanalyzer (Agilent, 2100), the barcoded libraries were pooled for single-end 150-bp sequencing in an Illumina Hiseq 2500 machine