GSM5492033: Arabidopsis thaliana Col-0 seedlings White light (W2); Arabidopsis thaliana; RNA-Seq
RNA was extracted using RNeasy Plant Mini Kit (Qiagen cat. 74904) using RNase-Free DNase (Qiagen cat. 79254) following manufacturer instructions The sequencing library construction was adapted from 3′-end RNA-seq protocol. The mRNA was fractionated from 20 µg of total RNA using Dynabeads Oligo (dT)25 (Invitrogen), and fragmented using Fragmentation Reagents (Ambion) at 70°C for 2.5 min in 20 µl of reaction. The polyA-tailed 3′-end fragments were captured by another run of mRNA purification as described above, and then treated by Antarctic Phosphatase (NEB) and T4 Polynucleotide Kinase (NEB) at 37°C for 1 h and 2 h, respectively. The sample was purified using RNeasy MinElute Cleanup Kit (Qiagen) according to Illumina's protocol. The eluted mRNA fragments were ligated with 2.5 µM of Illumina's SRA 5′ adaptor by T4 RNA Ligase 1 (NEB) at 20°C for 4 h. The 3′ cDNA adapter derived from Illumina's v1.5 sRNA 3′ adapter was conjugated with the anchored oligo (dT)20 primer, and introduced through reverse transcription using the SuperScript III First-Strand Synthesis System (Invitrogen). The first-strand cDNA was purified using the Agencourt AMPure XP system, and then amplified by PCR reaction using Phusion High-Fidelity DNA Polymerase with Illumina's sRNA PCR primer set. The size of purified library DNA was validated by Bioanalyzer 2000.