Salinity-induced transcriptional and DNA methylation reprogramming of peanut roots (Arachis hypogaea L)
Salinity-induced transcriptional and DNA methylation reprogramming of peanut roots (Arachis hypogaea L)
Roots of 4-5 peanut seedlings were pooled and considered as one biological replicate , peanut cv. GG 20 seeds were surface sterilized with 2% sodium hypochlorite and rinsed 4-5 times with sterilized double-distilled water. Seeds were placed in sterile cotton (soaked in ½ MS medium) in a tissue culture bottle and kept in the dark for two days before transfer to a growth chamber. The growth conditions were set at 26 °C and 16h/ 8h light/dark cycle (350 µmol m-2 s-1 light intensity) and seedlings were allowed to grow for 7 days.  Two-weeks old peanut seedlings were subjected to 150 mM NaCl (final concentration in hydroponics growth medium) and root samples were collected after one week of salt treatment.  50-100 ng of purified genomic DNA were treated with Zymo CT conversion reagent in a thermal cycler with defined parameters. The bisulphite treated DNA was purified and used to prepare sequencing library using True Seq Methylation kit  Genomic DNA was isolated from the root samples using CTAB method, followed by RNAse A treatment.50-100 ng of purified genomic DNA were treated with Zymo CT conversion reagent in a thermal cycler with defined parameters. The bisulphite treated DNA was purified and used to prepare sequencing library using True Seq Methylation kit (Illumina, Inc. USA). , bisulphite-treated single stranded DNA was random primed using a polymerase that can read Uracil. The 3' end of newly synthesized DNA strands were tagged with a second specific sequence followed by addition of Illumina adapters at both the DNA strands. The libraries were quantified with Qubit 3.0 and analyzed in 4200 Tape Station System (Agilent Technologies) using high sensitivity D1000 screen tape as per the manufacturer's instruction.