nanoCAGE-2000 24h Root Libraries (Experiment 3)
NanoCAGE-2000 samples in 9-day old Arabidoopsis thaliana roots
Tissue was ground in liquid N2 and RNA extracted with TRIzol reagent (Invitrogen; http://www.lifetechnologies.com). Total RNA (RIN 9.0 to 9.6) was treated with 1X RNA Secure Reagent (Ambion; http://www.lifetechnologies.com) at 65oC for 10 min, treated with DNase I (Ambion; http://www.lifetechnologies.com) for 10 min at 37oC, and purified with RNAeasy kit (Qiagen; http://www.qiagen.com) following the manufacturer’s instructions. rRNA was depleted with Ribo-Zero Magnetic kit (Plant Seed/Root) (Epicentre; http://www.epibio.com) following the manufacturer’s instructions. Purity and concentration of the resulting mRNAs was determined using Bioanalyzer (Agilent Technologies; http://www.agilent.com/home).  RT was performed as in (Salimulah et al. 2011) except that either 40 µl reactions with ~200 ng mRNA (Exp. 1 and 3), or 20 µl reactions with 50 ng mRNA + 100 ng total RNA were performed. Reactions were purified with Agencourt RNAClean XP kit (Beckman Coulter; https://www.beckmancoulter.com) as per the manufacturer’s instructions, and 1st strand cDNA eluted from the beads with 80 µl H2O. The optimal number of semisuppressive PCR cycles, usually between 17 and 25, required for second-strand synthesis were determined by quantitative real-time PCR as in (Salimulah et al. 2011).  No-template controls were included at this step to test for potential contaminants.  Next, for each library, 400 µl semisuppressive PCR reactions were performed using 60 µl of 1st strand cDNA and the selected number of cycles, after which the reactions were purified with Agencourt AMPure XP kit (Beckman Coulter; https://www.beckmancoulter.com). Concentration of PCR products was determined with Qubit dsDNA HS Assay kit (Molecular Probes; http://www.lifetechnologies.com), and products diluted to 10 ng/µl.  For each library, addition of sequencing adaptors was performed in 700 µl PCR mixtures. At this step, the ExTaq polymerase was replaced with Phusion Hot Start II High-Fidelity DNA Polymerase (Thermo Scientific; http://www.thermoscientificbio.com) in order to generate blunt cDNA ends. The cycling conditions were: 1) 98oC for 1 min, 2) 1 cycle of 98oC for 15 sec, 55oC for 10 sec, 68oC for 2 min, 3) 9 cycles of 98oC for 15 sec, 65oC for 10 sec, 68oC for 2 min. After completion of the PCR, remaining primers were purified by Exonuclease I digestion as in (Takahashi et al. 2012). Namely, 5 µl of Exo I (20 U/ µl) (New England Biolabs; https://www.neb.com) was added per 700-µl PCR mixture and the mixtures incubated at 37oC for 30 min. Each 700 µl PCR mixture was then mixed with 3.5 ml (5V) of PB buffer, purified by running through a single column of QIAquick PCR purification kit (Qiagen; http://www.qiagen.com), and each library eluted with 25 µl H2O.  Library concentrations were determined with Qubit dsDNA HS Assay kit (Molecular Probes; http://www.lifetechnologies.com), and library molecular size distributions determined using Bioanalyzer. The optimal amounts of libraries were then determined as per the Illumina qPCR quantification guide (Illumina; http://support.illumina.com), and libraries sequenced at concentrations of 1.3 to 2.3 nM.
S5-10BPLA
Oregon State University
nanoCAGE-2000 24h Root Libraries (Experiment 3)