GSM8366637: Seeds, Col-0, 18d after pollination, Batch A, rep2; Arabidopsis thaliana; RNA-Seq
Total RNA was extracted using a hot borate method modified from Wan and Wilkins (1994). The homogenized seeds were mixed with an extraction buffer (0.2 M Na borate decahydrate (Borax), 30 mM EGTA, 1% SDS, 1% Na deoxy-cholate (Na-DOC)) containing 1.6 mg DTT and 48 mg PVP40 which had been heated to 80ºC. One mg proteinase K was added to this suspension and incubated for 15 min at 42ºC. After adding 64 μL of 2 M KCl the samples were incubated on ice for 30 min and subsequently centrifuged for 20 min at 12,000 g. 270 μL of ice-cold 8 M LiCl was added to the supernatant in a final concentration of 2 M and the tubes were incubated overnight on ice. After centrifugation for 20 min at 12,000 g at 4ºC, the pellets were washed with 750 μL ice-cold 2 M LiCl. The samples were centrifuged for 10 min at 10,000 g at 4ºC and the pellets were re-suspended in 100 μL DEPC treated water. The samples were cleaned with phenol chloroform and treated with DNAse (RQ1 DNase, Promega). RNA was processed for RNA-seq using mRNA enrichment polyA capture (Illumina). The RNA-seq library was prepared for sequencing with strand-specific cDNA synthesis using random hexamer priming.