ChIP-Seq analysis for Lr28 mediated leaf rust resistance
Antibodies specific for H3K4Me3 and H3K27Me3 marks were procured from Abcam, USA.  The workflow for ChIP and preparation of ChIP-seq libraries included the following steps:  (i) chromatin was isolated (using Digenode Plant ChIP-seq kit) from all the eight samples (as above); (ii) isolated chromatin was sheared and immuno-precipitated using each of the above two antibodies, thus making 16 samples;  input DNA (total genomic DNA used as control) was also isolated from the above four samples (without biological replicates) following precipitation of chromatin using Digenode Plant ChIP-seq kit; in this manner 20 samples were available. (iii) DNA was isolated from all the 20 ChIPed samples and was used for constructing ChIP-Seq libraries using Microplex Library Prep Kit V2; (iv) Quantity and quality check (QC) of each amplified library was done by Bioanalyzer2100 (Agilent Technology) using High Sensitivity (HS) DNA chip based on Qubit concentration and mean peak size; (v) sequencing of 20 DNA libraries was outsourced to Xcelris Genomics Pvt. Ltd, Ahmedabad, who used Illumina platform for cluster generation and sequencing; paired-end (PE) sequencing (2x75 bp) was undertaken, which allowed template fragments to be sequenced in both forward and reverse directions. Sequencing data was generated with ~11-90 million reads/sample. Data in FASTQ files for all the 20 libraries was subjected to detailed bioinformatics analysis