title: RNA-seq of Glycine max: shoot
design: Soybean (Wm 82) was grown under white light condition (300 µmol•m-2•s-1) with a 12 h light/12 h dark circadian rhythm for 2 weeks. Shoots and roots were collected separately and used for NanoCAGE library construction. RNA extraction was conducted by using RNA extraction Kit (DP441, TIANGEN, China) by following the manufacturer's instructions. RQ1 DNase (M6101, Promega, USA) was added to remove DNA. NanoCAGE-seq libraries were prepared from 1 µg of total RNA of each sample. The integrity of RNA was further verified by 1.0% agarose gel electrophoresis. The total RNA was treated with T4 polynucleotide kinase (M0201L, NEB, USA) and subsequently digested with Terminator™ 5´-Phosphate-Dependent Exonuclease (TER51020, Epicentre, USA) to enrich the capped mRNA. Next, reverse transcription (RT) primer harboring a 3'-adaptor sequence and randomized hexamer was used to conduct RT. Subsequently, the 5' adaptor, which contained three additional guanine residues (rG) at its 3' terminus, was added to the RT reaction. The mixture was then incubated for another 30 minutes to facilitate template switching and the tagging of the 5' adaptor. The ligated products were amplified by PCR and purified, quantified and stored at -80 ℃ before sequencing. The NanoCAGE libraries were sequenced using the Illumina Novaseq Xplus system with 150 bp paired-end sequencing.