DNA-seq of Arabidopsis thaliana: chs5 mutant
Fresh material was ground with 800L of grinding buffer and incubated for 30 minutes at 65C. 700L of chloroform/isoamyl alcohol (24:1, v:v) were added and mixed by inverting the tubes. The supernatant was recovered after a centrifugation step at 10,000 rpm, 16C for 5 minutes. 50L of RNAse (10mg/mL) were added, and samples were incubated at 37C for 45 minutes. Once again, 700L of chloroform/isoamyl alcohol (24:1, v:v) were added to the samples and mixed. The supernatant was recovered after centrifugation and mixed with 1/2 volume of NaCl 5M and three volumes of cold ethanol, stored at -20C for one hour. DNA was pelleted by centrifugation at five 500rpm, 16C, washed three times with 1mL ethanol 70 and dried at room temperature before resuspension in 20L of water. At this stage, samples from reciprocal crossings and in the same phenotypical category were pooled together, and DNA was quantified using QubitTM (Thermo Fischer Scientific).