title: GSM1174499: T-DNA root; Arabidopsis thaliana; RNA-Seq
library_construction_protocol: Total RNA of 20-day-old shoot and root were extracted with DNase I digestion according to the manufacturer’s instructions (RNeasy Plant Mini Kit, Qiagen, Hong Kong). The preliminarily quality was detected by running 1% (w/v) agarose gel stained by GelRed (Biotium, USA). The quality of RNA samples was tested with Agilent 2100 Bioanalyzer RNA Nanochip. At least 10 µg of total RNA at a concentration of ≥ 400 ng/µl, OD260/280=1.8~2.2 were used for cDNA library preparation. Total RNA was sent to Beijing Genomics Institute (BGI) (Shenzhen, Guangdong province, China) and small RNAs were sequenced using Illumina HiSeqTM 2000 high throughput sequencing technology. SRNAs with the length of 18-30 nt were first separated from total RNA by running on 15% (w/v) TBE urea polyacrylamide gel. The excised gel with sRNAs from 18 to 30 nt in length were submerged in 600 μL of 0.3 M sodium chloride overnight at 4℃. The sRNA fragments were dissolved in diethylpyrocarbonate treated water (Ambion, Austin, TX, USA). The sRNAs were then added 5’ adaptor by T4 RNA ligase (Takara, Dalian, China) in the presence of RNase OUT Recombinant Ribonuclease Inhibitor (Invitrogen, China) overnight at 4℃ according to the manual. After obtaining the 5’ ligation products, a 3’adaptor was also added to the selecting 5’adaptor sRNAs following the same procedures as the 5’adaptor ligation. Finally, both 3’ and 5’ adaptors ligated sRNAs were run on a 10% (w/v) TBE urea polyacrylamide gel and then the 44 nt RNAs were excised. The sRNA were reversely transcribed to cDNA with the RT primer by Superscript II reverse transcriptase (Invitrogen, China). The cDNA was further quantified by Agilent 2100 and sequenced by the Illumina 1G Genome Analyzer.