title: GSM1847409: Treatment; Nicotiana benthamiana; ncRNA-Seq
library_construction_protocol: Our protocol for the purification of HCPro tagged with six histidines (6x-HCPro) is similar to the one described by Tena et al. (2013) with minor modifications.  Briefly, fully developed leaves showing symptoms were collected at seven days post-agroinoculation of plants with either the PVX or with the PVX-P1-6x-HCPro vectors.  Around 100 g of systemic infected tissue were collected and kept frozen until the time of use.  Every step in the purification protocol was performed between 0º and 4º C degrees with pre-chilled buffers and laboratory equipment.  Leaves were homogenized in extraction buffer [3.3 ml of buffer/g of leaf tissue: 100 mM Tris-HCl pH 8.5, 20 mM MgSO4, 500 mM NaCl, 0.5 mM, ethyleneglycol-bis(2-aminoethyl ether)-N,N,N',N′-tetra acetic acid (EGTA), 20% sucrose, supplemented with 0.2% Na2SO3, 0.1% polyvinylpyrrolidone-40 and 5 mM 2-mercaptoethanol]. After filtration, centrifugation and ultracentrifugation, total soluble proteins were precipitated with 40% (NH4)2SO4. Proteins were resuspended in extraction buffer and incubated with 1ml of nickel-nitrilotriacetic acid agarose (Ni-NTA Agarose, Qiagen GmbH, Hilden, Germany).  Several rinses of the resin were performed with extraction buffer and after that two consecutive steps of elution were made with 4 ml of extraction buffer supplemented with 400 mM EGTA.  The first elution contained more contaminant proteins from the host, such as rubisco.  The second elution was largely free from host proteins and contained the highest amount of eluted 6x-HCPro.Total RNA was extracted from 1.5 ml of the second elutions of purified samples obtained from tissues infected with either PVX-P1-6x-HCPro (hereafter the HCPro sample) or with PVX (hereafter the control sample). Total RNAs were isolated with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the instruction of the manufacturers.  For visualization, total RNAs were size fractioned by electrophoresis in a 1.5% agarose gel.  For deep-sequencing of the RNA population of 500 nts in length or less, total RNA were fractioned by electrophoresis in 10%-PAGE containing 8M urea.  The gel area from ~12 nts to ~500 nts was sliced with a razor and RNA was eluted from the gel with 0.3 M NaCl, and precipitated with 1 ul of glycogen (20 ug/ul) (Roche, Basel, Switzerland) and 1 volume of isopropanol. RNA sequencing was performed by Fasteris SA (Chemin du Pont-du-Centenaire, Genève, Switzerland). Briefly, RNA samples were subjected to 5' cap removing with tobacco acid pyrophosphatase (TAP) treatment and ends repair reactions, followed by single-stranded ligation of 3’ and 5’ indexed adapters.  Afterwards, reverse transcription and PCR amplification were performed to generate the two cDNA libraries (one from the control and another from the HCPro samples) that were multiplexed and sequenced in one lane of a 125 bp single-end run, using Illumina HiSeq 2500 sequencer (Illumina, San Diego, California, USA). RNA-seq 125 bp single-end run, using Illumina HiSeq 2500 sequencer (Illumina, San Diego, California, USA).
attributes.tag: GEO Accession
attributes.value: GSM1847409