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The SNP analysis used the DNA samples of five olive genotypes (Siyah Salamuralık, Yun Celebi, Yuvarlak Celebi, Hirhali Celebi and Halhali 3) that originated from different locations in Turkey. Total RNA was extracted using the RNeasy Plant Mini Kit (QIAGEN, CA, USA, Cat. Number: 74903). The poly (A) mRNA was purified from the total RNA using the Oligotex mRNA Midi Prep Kit (QIAGEN, Cat Number: 70022) followed by repurification using the mRNA-Seq-8 Sample Prep Kit (Illumina Inc. San Diego, USA, Cat. No: # RS-100-0801). The poly-A containing mRNA was purified from 2 µg total RNA using oligo(dT) magnetic beads and fragmented into 200–500 bp pieces using divalent cations at 94°C for 5 min. The cleaved RNA fragments were copied into first strand cDNA using SuperScript II reverse transcriptase (Life Technologies, Inc.). After the second strand cDNA synthesis, the fragments were end-repaired and a-tailed and the indexed adapters were ligated. The products were purified and enriched by PCR to create the final cDNA library. These pooled libraries were sequenced at the DNA Link, Inc. in Seoul, South Korea using an Illumina GAIIx (Illumina Inc., San Diego, CA, USA). The raw sequencing data were transformed by base calling into sequence data (i.e., raw data or raw reads) stored in FASTQ format. Next, we used cutadapt (https://code.google.com/p/cutadapt/) to remove any reads that were contaminated with an Illumina adapter. Then, the low-quality score regions and reads shorter than 70 bp were removed using our in-house script. In addition, a comprehensive ribosomal RNA database, the SILVA DATABASE (DB), containing regularly updated, high-quality sequences of eukaryotic rRNAs was incorporated into the cleaning pipeline to remove ribosomal RNA sequences. Reads that mapped to SILVA DB sequences were assumed to be ribosomal RNA and were removed. The resulting non-mapped reads were then considered to be mRNA. These cleaned mRNA reads were assembled using ABySS tools. The assembled contigs were reassembled using the de novo assembly tool Newbler version 2.3 (GS de novo assembler, Roche Applied Sciences). The cleaned mRNA reads (reads that did not map to SILVA DB sequences) were then mapped to Newbler’s output contigs, which were used as reference sequences. |