GSM2060146: F4_drought stress_Desiree; Solanum tuberosum; RNA-Seq
Homogenized leaf material of two to four replicates per cultivar and treatment was pooled to approximate 100 mg. Total RNA was isolated using a TRIzol protocol based on the “single step” method (Chomczynski and Sacchi 1987). For extraction 1 mL TRIzol reagent (Invitrogen) and 200 µL chloroform were used. The supernatant was precipitated by adding 500 µL isopropanol and 250 µL 2 M NaCl. After washing the pellet twice with 70% ethanol, it was re-suspended in 50 µL RNase-free water. Samples were transferred to the BGI in China for cDNA library preparation and sequencing. The total RNA samples are first treated with DNase I. Then the mRNA is enriched by using the oligo(dT) magnetic beads. Mixed with the fragmentation buffer, the mRNA is fragmented into short fragments. Then the first strand of cDNA is synthesized by using random hexamer-primer. Buffer, dNTPs, RNase H and DNA polymerase I are added to synthesize the second strand. The double strand cDNA is purified with magnetic beads. End reparation and 3'-end single nucleotide A (adenine) addition is then performed. Finally, sequencing adaptors are ligated to the fragments. The fragments are enriched by PCR amplification. During the QC step, Agilent 2100 Bioanaylzer and ABI StepOnePlus Real-Time PCR System are used to qualify and quantify of the sample library. The library products are ready for sequencing via Illumina HiSeqTM 2000.
GEO Accession
GSM2060146