title: GSM3181995: ROOT ZONE I (0.5mm) REP 1 [RNA-seq]; Arabidopsis thaliana; RNA-Seq
library_construction_protocol: Total RNA was extracted from these frozen samples using Picopure RNA isolation kit (Arcturus®, Life technologies). RNA concentration was determined on a Qubit (Thermo Fisher/Life Technologies, Inc) and sample quality was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Inc.). Library preparation:  Five ng of total RNA was used for cDNA library construction using CloneTech SMART-Seq V3 ultra low input RNA kit for sequencing (Clontech Laboratories, Inc,) according to manufacturer's protocol. Briefly, 1st strand cDNA was primed by the SMART-SMARTer II A oligonucleotide and then base-pairs with these additional nucleotides, creating an extended template. The reverse transcriptase then switches templates and continues transcribing to the end of the oligonucleotide, resulting full-length cDNA that contains an anchor sequence that serves as a universal priming site for second strand synthesis. cDNA was amplified with primer II A for 10 PCR cycles. Then Illumina sequencing libraries were generated with 120 pg of cDNA using Illumina Nextera DNA Sample Preparation Kit (Cat#: FC-131-1024) per manufacturer's instructions. Briefly, 120 pg of cDNA was fragmented by tagmentation reaction and then adapter sequences added onto template cDNA by PCR amplification. Libraries were quantitated by Bioanalyzer and qPCR (Kapa Biosystems, catalog number: KK4824). Finally, the libraries were pooled by equal molar concentration and sequenced by Illumina 2X150 NextSeq 500 (Illumina Inc., CA, U.S.). Illumina sequencing:  Sequencing experiments were performed at the Interdisciplinary Center for Biotechnology Research (ICBR) gene expression and sequencing core, University of Florida. In preparation for sequencing, barcoded libraries were sized on the bioanalyzer, quantitated by QUBIT and qPCR (Kapa Biosystems, catalog number: KK4824). Individual samples were pooled equimolarly at 4 nM. This “working pool” was used as input in the NextSeq500 instrument sample preparation protocol (Illumina, Part # 15048776, Rev A). Typically, a 1.3 pM library concentration resulted in optimum clustering density in our instrument (i.e., ~200,000 clusters per mm2). Samples were sequenced on a single flowcell, using a 2x150 cycles (paired-end) configuration. A typical sequencing run in the NextSeq500 produced 750-800 million paired-end reads with a Q30>=85%. For RNA seq, around 40 million reads/sample provided sufficient depth for transcriptome analysis.