title: GSM9322976: BD_Before_Drought; Triticum aestivum; Bisulfite-Seq
library_name: GSM9322976
library_construction_protocol: Each DNA extraction consisted in 6cm of leaf tissue. BD tissue was collected upon the wheat seedlings reaching GS13 whilst the AD tissue was collected at the end of the drought period (13 days after GS13, 10 days draught stress+3 days recovery). Tissue was collected individually for each sample, and immediately immersed in liquid nitrogen to prevent nucleic acid degradation. Tissue samples were stored at -80°c for later processing. Genomic DNA was extracted from <100 mg of individual leaf tissue samples using the the Wizard HMW DNA Extraction Kit (Promega, WI, USA) according to the manufacturer's protocol, including an RNAse treatment. DNA was initially measured using NanoDrop ND-1000 Spectrophotometer (Thermo-Fisher Scientific, MA, USA) and a Qubit 4 Fluorometer (Life Technologies, CA, USA) whilst the quality was assessed by running 2 μl of sample on an Agilent Technology Tapestation (Agilent Technologies, CA, USA) and only keeping samples with DIN>7. Replicates (4 plants) were pooled into 1 sample per landrace per treatment at equimolar proportions. Each pool was pooled again (7 landraces) per treatment in equimolar proportiopns. The library was constructed as thus: After the addition of lambda DNA negative control, DNA samples were fragmented into 200-400bp using Covaris S220. DNA fragments were repaired, dA tailed, and ligated to sequencing adaptors with all methylated cytosines. DNA fragments were bisulfite treated with the EZ DNA Methylation Gold Kit (Zymo Research). After the treatment, Cytosines without methylation changed to U (after PCR amplification to T), while Cytosines with methylation remained unchanged. The library was then subject to size selection and PCR amplification.
identifiers.namespace: GEO
identifiers.id: GSM9322976_r1