GSM8443458: WL_2; Malus spectabilis; RNA-Seq
Total RNA was isolated from each sample using a previously reported method modified by the addition of a purification step, and cDNA was synthesized as previously described (Xia et al., 2012).Xia, R., Zhu, H., An, Y. Q., Beers, E. P., and Liu, Z. (2012). Apple miRNAs and tasiRNAs with novel regulatory networks. Genome Biol. 13:R47. doi: 10.1186/ gb-2012-13-6-r47 After treatment with DNase I to eliminate contaminating DNA, mRNA was purified from total RNA extracts using magnetic oligo (dT) beads. The mRNA was mixed with fragmentation buffer and cleaved into short fragments for use as templates for cDNA synthesis. Short fragments were purified, resolved with buffer for end reparation and single adenine nucleotide addition and connected with adapters. After agarose gel electrophoresis, suitable fragments were selected as templates for PCR amplification. An Agilent 2100 Bioanalyzer and an ABI StepOnePlusTM Real-Time PCR system were used for quantification and quality assessment of sample libraries. The constructed libraries were sequenced using an Illumina HiSeqTM 2000 system (BGI, Shenzhen, China).