GSM4103521: Shepody before rep2; Solanum tuberosum; RNA-Seq
Sampling was done with freshly harvested tubers (within two weeks of harvest) before cold storage and tubers after cold storage.   Each tuber was sampled once at either end and three times around the middle for a total of five cores per tuber in regions without eyes. For each replicate the cores from three tubers were pooled in 50 ml Falcon tubes. This results in 15 cores from three tubers per replicate.  Three replicates were taken from each clone for a total of nine tubers per clone. Tuber cores were flash frozen in liquid nitrogen and freeze-dried. Two 5/32” stainless steel balls (MP Biomedicals) were added to tubes containing tuber cores and the tuber cores were ground to a powder using Geno/Grinder (SPEX, Metuchen, U. S. A.) at 500(1X), for 2 minutes. Freeze-dried tuber powder was then stored at room temperature. The same tuber powder was used for both glucose determination and RNA extraction. RNA extraction from freeze-dried tuber tissue was performed using an Agencourt RNAdvance Tissue kit with  modifications (Tai et al. 2013). One ml Hot Borate buffer (200mM sodium borate decahydrate (Borax) pH 9.0, 30mM EGTA, 1% (w/v) SDS, 1% (w/v) sodium deoxycholate, and, added just before use, 10mM dithiothreitol and 2%(w/v) PVP (MW 40,000)) were added to 150 mg freeze-dried tuber core powder and the sample was homogenized using a vortex. The samples were centrifuged for 5 minutes at 10,000rpm and 200μl of the supernatant was transferred to a 96-well plate. The remaining sample was frozen at -80°C. To the samples in the 96-well plate 10μl of Agencourt proteinase K solution was added and the samples were mixed gently by pipetting up and down. Following addition of Agencourt proteinase K solution the samples were incubated at 37°C for 25 minutes. At this point the protocol followed the standard automated Agencourt RNAdvance Tissue kit with the Beckman Counter BioMek NXp workstation. Libraries were generated using the TruSeq RNA kit (Illumina). Messenger RNA was purified from 1 mg of total RNA using oligo-dT beads. The mRNA enriched fraction was reverse transcribed to generate cDNA fragments that were sheared to yield ~200 bp fragments. Following end-repair, 3' end adenylation steps, and index ligation, a PCR amplification step was performed. There were three biological replicates for each of the cultivars generating a total of 30 samples. A separate index was used for each cultivar-replicate combination. The quality of the library was assessed on a DNA 1000 chip and quantified by qPCR. Libraries were subjected to 100 bases of sequencing on a HiSeq 2000 (Illumina) instrument in paired-end mode. Initial quality control of the data was performed using the software included with the sequencer.