Apple transcriptome during superficial scald development in 'Granny Smith',
Strand specific RNAseq library construction - Transcriptomic profiling was performed on 3 composite (3 fruit each) biological replications for each orchard/time point combination using the protocol described by Gapper et al [14].  Briefly, total RNA was extracted using a chloroform phase extraction and purified using a RNAeasy column (Qiagen),.RNA-seq libraries were created using total RNA according to Zhong et al [94]. mRNA was isolated from total RNA, fragmented and used as a template for cDNA synthesis via reverse transcription (Superscript III, Invitrogen).  After the first strand of cDNA synthesis, the second strand was synthesized via a dNTP mix utilizing dUTP instead of dTTP. Ends of the double-stranded  cDNAs were then repaired (Enzymatics), dA tailed by the Klenow enzyme (Enzymatics), and universal TruSeq adapters (Illumina) were ligated. After ligation, the second strand was digested by uracil DNA glycosylase (UDG) to produce strand-specific enrichment of the library.  The UDG-digested cDNA was then used as a template to enrich the libraries by polymerase chain reaction (PCR) using primers containing the TruSeq barcodes, and the high-fidelity enzyme Phusion (NEB) with the conditions described in Gapper et al [14]. 20 ng of the libraries were pooled for sequencing; 48 were multiplexed per sequencing reaction using an Illumina HiSeq 2000/2500 next-generation sequencer at the Weill Medicine School Sequencing Facility (Cornell University, New York City, NY, USA).
4MDPArep3