2.4. RNA sequencing and bioinformatic analyses
RNA integrity number (RIN) was measured using Agilent 2100 bioanalyzer
(Palo Alto, CA, USA) and samples with RIN of 7.5 or above were sent to
CnK genomics for sequencing. Libraries were prepared using the Illumina
TruSeq Stranded mRNA sample preparation kit (San Diego, CA, USA), and a
total of six libraries were constructed using an Illumina Nexseq 500
platform (San Diego, CA, USA). Raw reads were trimmed by filtering out
adaptors with a minimum length of 75 bp using Trimmomatic v0.36
(http://www.usadellab.org/cms/index.php?page=trimmomatic) (Bolger et
al., 2014). The quality score was evaluated using fastqc
(http://www.bioinformatics.babraham.ac.uk/projects/fastqc) both before
and after trimming. Trimmed reads were aligned to apple reference genome
GDDH13 v1.1 (http://www.iris.angers.inra.fr/gddh13) using HISAT2
software (Kim et al., 2015). Across all the libraries, gene counts were
calculated for each predicted coding DNA sequence using FeatureCounts
v1.5.2. Read counts in the range of 21,543,441 to 24,876,113 were
obtained with Q30 ratio > 0.9. Reads per kilobase per
million (RPKM) values were counted from BAM files. Differentially
expressed genes (DEGs) with a false discovery rate (FDR) value of
< .05 and |log2 fold change| >
1 were selected using EdgeR Bioconductor software (Empirical analysis of
digital gene expression data in R) (Robinson et al., 2010). Functional
enrichment analysis of DEGs was performed using InterPro2
(http://www.ebi.ac.uk/interpro) and SwissProt (www.ebi.ac.uk/swissprot).
All sequencing data were deposited in National Center for Biotechnology
Information Sequence Read Archive database bearing the BioProject ID
PRJNA613278.