title: Illumina HiSeq 2500 paired end sequencing
design: We assessed the extent of salt-induced alterations of DNA methylation in barley. Using methylation-sensitive GBS (msGBS), we screened the leaf and root methylomes of five divergent barley varieties grown under control and three salt concentrations, to seek genotype independent salt-induced changes in DNA methylation
library_construction_protocol: After four daily exposures to 0mM NaCl beginning at Day 10, leaf sheath samples were collected from the barley strain Yarra and snap frozen in liquid nitrogen. Genomic DNA was digested using the combination of a rare cutter, EcoRI (GAATTC) and a frequent, methylation sensitive cutter MspI (CCGG). Each sample of DNA was digested in a reaction volume of 20microL, containing 2microL of NEB Smartcut buffer, 8U of HF-EcoRI (High-Fidelity) and 8 U of MspI (New England BioLabs Inc., Ipswich, MA, USA). The reaction was performed in a BioRad 100 thermocycler at 37C for 2 h, followed by enzyme inactivation at 65C for 10 min. DNA samples were allocated to plates (81 samples each), including the negative control, water. Prior to pooling plate samples into a single 81-plex library, the ligation products were individually cleaned up to remove excess adapters using an Agencourt AMPure XP purification system (#A63880, Beckman Coulter, Brea, CA, USA) at a ratio of 0.85, following the manufacturer's instructions. Individual GBS libraries were produced by pooling 25 ng of DNA from each sample. Each constructed library was then amplified in eight separate PCR reactions (25microL each), containing 10microL of library DNA, 5microL of 5x Q5 high fidelity buffer, 0.25microL of polymerase Q5 high fidelity, and 1microL of each Forward and Reverse common primer at 10microM, 0.5microL of 10microM dNTP and 7.25microL of pure sterile water. PCR amplification was performed in a BioRad T100 thermocycler (BioRad, Hercules, CA, USA), consisting of DNA denaturation at 98C (30 s) and ten cycles of 98C (30 s), 62C (20 s) and 72C (30 s), followed by 72C for 5 min. PCR products were next pooled to reconstitute libraries. DNA fragments between 200 and 350 bp in size were captured using Agencourt AMPure XP magnetic beads following the manufacturer's instructions. Bead-captured fragments were eluted in 35microL of water, and 30microL of the elution was collected in a new labelled microtube. Next, libraries were 125 bp paired-end sequenced in an Illumina HiSeq 2500 platform (Illumina Inc., San Diego, CA, USA) at the Australian Genome Research Facility (AGRF, Melbourne node, Australia).