Pe cluster kit v3 cbot hs

RNA (~3 μg per sample) was used as the input material for constructing libraries. RNA-seq libraries were prepared using the TruSeq Paired-End (PE) Cluster Kit v3-cBot-HS (Illumina, PE125) according to the manufacturer's protocol. Libraries from fertile and sterile flowers, with three biological replicates, were sequenced in a single Illumina Hiseq 2500 flowcell, generating >139 million paired-end reads per sample. A Perl script was written to remove low-quality sequences (reads with a base quality < 20). For de novo reference transcriptome assembly, all high-quality RNA-Seq reads were pooled from the Illumina sequencing of each of the six samples (three biological replicates) and were then used as input for assembly using Trinity software (Grabherr et al., 2011 (link)). All raw sequence data have been deposited in the NCBI Sequence Read Archive (SRA, accession number SRP076665).

All samples were enriched for human mitochondrial DNA via bead capture hybridization as detailed elsewhere33 . After enrichment the libraries were amplified in 100 μl reactions with 15 μl template, 2 units Phusion High Fidelity DNA polymerase, 1 unit 5 × HF buffer, 0.25 mM dNTPs and 0.3 μM IS5 and IS6 primers22 , and the following thermal profile: 5-min initial denaturation at 95 °C, followed by 16–23 cycles consisting of 30-s denaturation at 95 °C, a 30-s annealing at 60 °C and a 45-s elongation at 72 °C and a 5-min final elongation at 72 °C. Subsequently, the libraries were purified and quantified as described before and paired-end dual index sequencing was carried out on an Illumina HiSeq 2500 platform by 2 × 100+7+7 cycles following the manufacturer's protocols for multiplex sequencing (TruSeq PE Cluster Kit v3-cBot-HS).