TSS-Seq (nanoCAGE-XL) library from root sample from Arabidopsis thaliana
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 manufacturers instructions. rRNA was depleted with Ribo-Zero Magnetic kit (Plant Seed/Root) (Epicentre; http://www.epibio.com) following the manufacturers 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 l reactions with ~200 ng mRNA (Exp. 1 and 3), or 20 l 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 manufacturers instructions, and 1st strand cDNA eluted from the beads with 80 l 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 l semisuppressive PCR reactions were performed using 60 l 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/l. For each library, addition of sequencing adaptors was performed in 700 l 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 l of Exo I (20 U/ l) (New England Biolabs; https://www.neb.com) was added per 700-l PCR mixture and the mixtures incubated at 37oC for 30 min. Each 700 l 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 l 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.