GSM1846375: MET_degradome; Solanum pimpinellifolium; OTHER
Total RNAs were extracted with Trizol reagent (Invitrogen, CA, USA), and the quantity and purity of the total RNA were detected using a Bioanalyser 2100 and RNA 6000 Nano LabChip Kit (Agilent, CA, USA).Leaf samples from LA2093 exposed to 26/18°C, 33/33°C and 40/40°C for 8 h were used to construct three sRNA libraries, which we called the NT, MET and AET libraries, respectively. The three sRNA samples were extracted using TruSeq Small RNA Sample Prep Kits (Illumina, San Diego, USA). The sRNAs were reverse transcribed to cDNAs after isolation and ligation. The purified cDNAs from three sRNA libraries were sequenced with Illumina Hiseq2500 (LC-BIO, Hangzhou, China).All samples were assigned RNA integrity number (RIN) values of at least seven. Approximately 20 µg of total RNAs were used to construct the degradome libraries. The NT, MET and AET degradome libraries were constructed according to the methods described by Addo-Quaye et al. (2008)23 and Ma et al. (2010)53, with minor modifications. The mRNA was mixed with biotinylated random primers, and then the RNA containing the biotinylated random primers was captured by beads and ligated to 5’ adaptors. First-strand cDNA was generated from the ligated sequence after reverse transcription. A number of DNA products were produced by PCR amplification. Following purification, digestion, ligation and repurification, the cDNA library was sequenced with an Illumina Hiseq2500 (LC-BIO, Hangzhou, China).