title: GSM1290201: cassava_stems_ORST4_7dpi_1; Manihot esculenta; RNA-Seq
library_construction_protocol: Samples collected were immediately submerged in liquid nitrogen and then stored at -80°C. 100mg of grounded tissue was incubated in lysis buffer (b-mercaptoethanol 2%, PVP, 2%, SDS 1%, EDTA 25mM, NaCl 100mM and tris HCl pH 7.5 100mM). RNA extraction was carried out with acid phenol:cloroform:isoamylic 25:24:1 and precipitated using  NaCl 5M and ethanol. RNA was treated with DNase I following the instructions of commercial manufacturer (Ambion). Once the DNA-free total RNA was obtained it was visualized by agarose gel electrophoresis and quantify in NanoDrop (Thermo Scientific). Protocol for library constriction was adapted from (Zhong et al. 2011). Six RNAseq libraries were constructed from 7ug of DNA-free mRNA purified by oligo dT25 Dynabeads (invitrogen).  cDNA was synthesized employing Superscript III Reverse Transcriptase (Life technologies). cDNA quality and quantification was done using an Agilent Bioanalyzer on a High sensitivity DNA chip (Agilent Technologies) and an equal amount (20-50 ng) of index libraries were pooled together. The libraries were sequenced in two different lanes of Illumina Sequence (technical replicates). Each library was Paired End (PE) sequenced.
attributes.tag: GEO Accession
attributes.value: GSM1290201