Truseq sbs kit v3

Quantification of Illumina sequencing libraries with KAPA Library Quantification Kit, normalization, and pooling were performed following standard protocols for sequencing in the Illumina MiSeq platform. Pooled libraries were subjected to 2–3 runs using the MiSeq Reagent kit v2 (500-cycle format, paired-end (PE) reads) and resulting sequences for each library were combined. RNA-seq libraries were also run on a single-sequencing lane on Illumina HiSeq2500 (200-cycle format) using TruSeq SBS v3 kits, and resulting sequences were combined with the MiSeq PE sequences. On average, Illumina PE read1 and read2 presented, respectively, >80% and >75% of bases with quality score at least 30 (Q30).
DNA samples purified from ZC3 chamber were also submitted to pyrosequencing following standard Roche 454 GS FLX Titanium protocols (Roche Applied Science) as previously described13 (link).

For each of the 14 samples (12 culms, 2 rhizomes), a polyA cDNA library was prepared from 4 μg of total RNA using a TruSeq Stranded mRNA LT Kit (Illumina, San Diego, CA, USA) following the manufacturer’s low sample (LS) protocol. A chemical fragmentation step of 30 s at 94°C as described in the Illumina protocol was used to prepare insert lengths between 130 and 340 bp with an aim of producing a final library size of c.450 bp. Fourteen complimentary adapters (Illumina) were chosen with the aid of a barcode diversity calculator¹ and ligated to the sample inserts. For each of the 14 cDNA libraries, the fragment size average and range was assessed using a Bioanalyzer and associated DNA1000 reagent kit (Agilent Technologies, Santa Clara, USA) and the concentration determined using a Qubit 1.0 fluorometer (ThermoFisher Scientific, Wilmington, USA).
The concentration of each cDNA library was normalized to 10 nM before being pooled for processing. Paired-end sequencing of the libraries was undertaken at La Trobe University (Melbourne, VIC, Australia) on a HiSeq^TM1500 platform after preparation with a TruSeq PE Cluster Kit v3-cBot-HS and a TruSeq SBS v3 kit (Illumina, San Diego, USA). The libraries were run across a proportion (c.74%) of two lanes on a flow cell.

Illumina deep sequencing as well as quantification of small RNA content were performed at a genomics core facility: Center of Excellence for Fluorescent Bioanalytics (KFB, University of Regensburg, Germany). For deep sequencing, all libraries were quantified using the KAPA SYBR FAST ABI Prism Library Quantification Kit (Kapa Biosystems, Woburn, MA, USA). Equimolar amounts of each library were used for cluster generation on the cBot with the TruSeq SR Cluster Kit v3 (Illumina, San Diego, CA, USA). The sequencing run was performed on a HiSeq 1000 instrument (Illumina, San Diego, CA, USA) using the indexed, 50 cycles single read (SR) protocol and the TruSeq SBS v3 Kit (Illumina, San Diego, CA, USA). Image analysis and base calling resulted in .bcl files which were then converted into .fastq files by the CASAVA1.8.2 software.