GSM9540903: dbr1-2_dcl2-1_TuMV_mRNA_R3; Arabidopsis thaliana; RNA-Seq
Arabidopsis tissue (0.4 g) was ground in liquid nitrogen with 1.25 ml cold PEB buffer (200 mM Tris-HCl, pH 9.0; 200 mM KCl; 35 mM MgCl2; 25 mM Na2EDTA; 5 mM DTT; 1 % Brij-35 ; 1 % IGEPAL® CA-630; 1 % Tween 20; 1 % Triton X-100; 1 % polyoxyethylene 10 tridecyl ether; 1 % DOC; 0.5 x Cocktail; 1 mM PMSF; 100 μg/ml CHX; 100 μg/ml Chl; 0.5 U / ml RRI, and 10 units/mL DNase I [Invitrogen AM2238]) and incubated on ice for 20 mins. After centrifugation twice at 16,000 g for 15 mins at 4 °C, lysate contains mainly monosomes which were layered onto a 12 ml 15 % to 60 % sucrose gradient, and centrifuged at 4 °C in a Beckman SW41Ti rotor at 35,000 r.p.m. for 7 h. Samples were peak fractionated by A260 absorption using the gradient station (Biocomp). polysome fractions were collected and subjected to RNA extraction with Trizol LS. For sRNA sequencing (sRNA-seq),we prepared sRNA libraries using the NEBNext Small RNA Library Prep Set for Illumina.For lariat RNA-seq, 20 μg of total RNA were incubated at 37°C for 1 h in 50 μL reactions with RNase R (Epicentre, USA) at a final concentration of 1 U RNase R per μg total RNA. Then, RNAs were purified using the RNA Clean & Concentrator-5 (Zymo Research) following the manufacturer's instructions. A Ribo-off kit (Vazyme, Nanjing, China) was used to remove rRNA from the above RNase Rtreated RNAs. 50 ng of RNA for each sample was fragmented with Frag/Prime Buffer (Vazyme) at 80°C for 1 min and used as the starting material for strand-specific RNA-seq library preparation using the VAHTS Universal V8 RNA-seq Library Prep Kit for Illumina (Vazyme). For lncRNAseq, 5 μg of total RNA for each sample was treated by the Ribo-off kit to remove rRNA and RNA. 50 ng of RNA for each sample was fragmentated at 94°C for 5 min for strand-specific RNA-seq library preparation using the above kit. For Ribo-seq,the ribosome footprint fragments were repaired via T4 PNK (NEB M0201S) in the absence of ATP and rRNA depletion was performed using Ribo-off kit (Yeason 12254ES). RNA 3' ligation, 5' ligation, reverse transcription and Library Amplification were performed using The QIAseq miRNA Library Kit (QIAGEN 331595). The PCR amplified cDNA construct was purified using a Monarch PCR & DNA Kit (NEB T1030S). The cDNA was separated via 6% (w/v) TBE-PAGE 10-well gel and and the library of the expected size (~180 bp) was selected and recovered.