title: GSM2879583: Sample 49_600_N-S+_rep1; Triticum monococcum; RNA-Seq
library_construction_protocol: RNA were extracted from einkorn grain according to the method used by (Pont et al., 2011). Three biological replicates were used. The 54 grain samples (three replicates at 100 and 200°Cd after anthesis and three replicates × four treatments [N-S-, N+S-, N-S+, N+S+] at 300, 400, 500 and 600°Cd after anthesis) were ground in liquid nitrogen and 4.5 mL of buffer (10 mM Tris-HCl, pH7.4, 1 mM EDTA, 0.1 M NaCl, 1% SDS) were added to approximately 0.5 g of flour. The supernatant was collected and 3 mL of a 25 : 24 : 1 phenol : chloroform : isoamyl alcohol mixture (v : v) were added to eliminate starch and proteins. Supernatant was collected, and nucleic acids were precipitated by adding 0.1 volume of 3M AcNA (pH5.2) and 2 volumes of 100% ethanol. The pellet was dried under ambient condition for 1 h and dissolved in RNase-free water. A DNAse treatment was then performed using a RNase-Free DNase set (Qiagen) and then a RNeasy MinElute Cleanup kit (Qiagen). RNA strand-specific libraries were created using commercially available kits according to the manufacturer's instructions. In brief, polyA-RNA was extracted from total RNA using an oligodT-bead based method. After fragmentation of the mRNA, first and dUTP based second strand synthesis was carried out, followed by end-repair, A-tailing, ligation of the indexed Illumina Adapter and digestion of the dUTP-strand. Size selection was done using a bead based method targeting an average insert size of 150-400 bp. After PCR amplification, the resulting fragments were cleaned up, pooled, quantified and used for cluster generation. For sequencing, pooled libraries were loaded on the cBot (Illumina) and cluster generation was performed using manufacturer's instructions. Paired-end sequencing using 125 bp read length was performed on a HiSeq2500 machine (HiSeq Control Software 2.2.38) using HiSeq Flow Cell v4 and TruSeq SBS Kit v4.
attributes.tag: GEO Accession
attributes.value: GSM2879583