GSM3502438
GSM3502438: PS_Drought; Phaseolus vulgaris; RNA-Seq
Pooled plant samples were powdered by grinding the frozen tissue. Each pool included 6-9 plants of each cultivar under control conditions or drought treatment. Thus, 12 RNA samples were extracted in two replicates under either drought or control conditions. Total RNA was extracted using 200 mL of the powdered sample and adding 700 mL of the Z6-extraction buffer (8 M guanidinium-HCl, 20 mM MES, 20 mM EDTA, 50 mM β-mercaptoethanol, pH 7.5). Then, an equal volume of phenol was added to carry out the extraction of RNA, followed by purification using the ssDNA/RNA Clean & Concentrator™ kit (Zymo Research Corp, Orange, CA, USA), according to the manufacturer's instructions. Equal amount of RNA from control and drought conditions were pooled together for further analysis, resulting in two RNA populations. The integrity of RNA was checked by agarose gel electrophoresis and the Agilent 2100 bioanalyzer (Agilent Technologies, Palo Alto, CA). The concentration of total RNA was determined using the NanoDrop ND-1000 spectrophotometer (NanoDrop, Wilmington, DE). Two libraries were constructed for each treatment using the TruSeq RNA Sample Preparation Kit (Illumina, Inc., San Diego, US-CA), following the manufacturer's recommendations. In brief, poly(A)-tailed mRNA was enriched and fragmented, followed by first cDNA synthesis. Subsequent second strand cDNA synthesis and the final reactions were cleaned up prior to perform the end repair step, and the addition of a single adenylate into the 3´ends. Adapters were ligated to both ends of the short fragments, which were enriched by 36 PCR cycles prior to be cleaned up and validated. cDNA fragments pools were loaded to Illumina MiSeq (Illumina, Inc., San Diego, US-CA) platform for single-ended sequencing.
GEO
GSM3502438
GEO Accession
GSM3502438