title: GSM4929911: Roots_Low Fe_3; Triticum aestivum; RNA-Seq
library_construction_protocol: Isolation of total RNA from the roots and flag leaves was carried out using the Isol-RNA Lysis Reagent (5 PRIME, USA). The total RNA was digested with RNase-Free DNase (Qiagen, Germany) to remove the genomic DNA followed by RNA cleaning with RNeasy Plant Mini Kit (Qiagen, Germany). The quality of the isolated RNA was determined with a Qubit® (1.0) Fluorometer (Life Technologies, USA) and a Bioanalyzer 2100 (Agilent, Germany). Strand-specific cDNA libraries (dUTP method) were prepared using TruSeq Stranded mRNA Sample Prep Kit (Illumina, USA). Total RNA samples (1 μg) were poly-A enriched and then reverse-transcribed into double-stranded cDNA with Actinomycin added during first-strand synthesis. The cDNA samples were fragmented, and end-repaired before ligation of TruSeq adapters containing the index for multiplexing fragments. After ligation, these TruSeq adapters were selectively enriched by PCR amplification on both ends. The quality and quantity of the enriched libraries were validated using Qubit® (1.0) Fluorometer and the Caliper GX LabChip® GX (Caliper Life Sciences, USA). The product is a smear with an average fragment size of approximately 250 bp. The libraries were normalized to 10 nM in 10 mM Tris-Cl, pH8.5 with 0.1% Tween 20. TruSeq PE Cluster Kit v3-cBot-HS (Illumina, USA) was used for cluster generation using 8 pM of pooled normalized libraries on the cBOT. Sequencing was performed on the Illumina HiSeq 2000 paired end at 2 × 101 bp (Illumina, USA).
attributes.tag: GEO Accession
attributes.value: GSM4929911