tissue: mature leaves age: 1.5 months after planting
Treatment protocol
None
Growth protocol
Stem cuts of sweet potato [I. Batatas (L.) Lam. cv. Xushu 18] were planted in June, 2009, and grown under normal conditions in Chengdu, Sichuan Province of China. Tissues of young leaves (YL), mature leaves (ML), and stems (Stem) were collected at 1.5 months after planting, and fibrous roots (FR), initial tuberous roots (ITR), expanding tuberous roots (ETR) and harvest tuberous roots (HTR) were collected at 1, 1.5, 3 and 5 months after planting, respectively.
Extracted molecule
total RNA
Extraction protocol
Total RNAs were extracted using the Trizol® Reagent (Invitrogen, USA), and treated with DNase I (Fermentas, USA) according to the manufacturer’s instructions. RNA integrity was checked using the Agilent 2100 Bioanalyzer (Agilent Technologies, USA). Double-stranded cDNA was synthesized using oligo (dT) beads. The cDNA was then digested with an anchoring restriction enzyme Nla III, which recognizes and cuts 3’ ‘CATG’. The digested cDNA samples were ligated to Illumina specific adapter A, containing a recognition site for enzyme Mme I. Following Mme I digestion, the Illumina specific adapter B containing a 2 bp degenerated 3’ overhangs was ligated. The adapter-ligated cDNA tag library was then enriched by PCR primers annealing to the adaptor ends. The PCR program was as follows: 30 s at 98 °C, followed by 15 cycles of 98 °C for 10 s, 60 °C for 30 s and 72 °C for 15 s, and then 72 °C for 5 min. After gel-purification, cDNA was used for cluster generation on separated flow cell lane. Sequencing by synthesis was performed using the Illumina Genome Analyzer II according to the manufacture’s protocol. Image analysis, base calling, extraction of tags and tag counting were performed using the Illumina pipeline.
Library strategy
RNA-Seq
Library source
transcriptomic
Library selection
cDNA
Instrument model
Illumina Genome Analyzer II
Description
Illumina DGE tag profiling.
Data processing
Counts: 3' adaptor sequence removal: since tags are only 21nt long while the sequencing reads are 35nt long, raw sequences are with 3' adaptor sequences; Empty reads removal (reads with only 3' adaptor sequences but no tags); Low quality Tags removal (Tags with unknown sequences ‘N'); Removal of Tags which are too long or too short, leaving Tags of 21nt long; Removal of Tags with a copy number of 1 (probably a sequencing error); Generate Clean Tags.
