title: GSM3891040: Ler#3; Arabidopsis thaliana; RNA-Seq
library_construction_protocol: Total RNA was extracted using plant RNA mini prep kit (ZYMO RESEARCH) RNA purity was determined by assaying 1 µL of total RNA extract on a NanoDrop8000 spectrophotometer (Thermo Scientific). Total RNA integrity was checked using an Agilent Technologies 2100 Bioanalyzer with an RNA integrity number value. mRNA sequencing libraries were prepared according to manufacturer's instructions (Illumina Truseq stranded mRNA library prep kit). mRNA was purified and fragmented from total RNA (1 ug) using poly-T oligo-attached magnetic beads and two rounds of purification. Cleaved RNA fragments primed with random hexamers were reverse transcribed into first-strand cDNA using reverse transcriptase, random primers and dUTP in place of dTTP. (The incorporation of dUTP quenches the second strand during amplification because the polymerase does not incorporate past this nucleotide.) A single adenine base was added to these cDNA fragments and was followed by adapter ligation. The products were purified and enriched with PCR to create the final strand-specific cDNA library. The quality of the amplified libraries was verified by capillary electrophoresis (Bioanalyzer, Agilent). After qPCR using SYBR Green PCR Master Mix (Applied Biosystems), the index-tagged libraries were combined in equimolar amounts within a single pool.