title: GSM4434546: Tomato_PSTVd_L2; Solanum lycopersicum; RNA-Seq
library_construction_protocol: Total RNA from infected leaf samples was extracted using the mirVana miRNA isolation kit (Ambion) with modifications. Briefly, 150 mg of leaf sample was ground with 600 µL of lysis/binding buffer in the presence of sand. Then, 70 µL of microRNA homogenate was added and the sample was mixed by vortexing followed by incubation on ice for 10 min. Total RNA was isolated by acid phenol-chloroform (5:1) extraction and was precipitated by centrifugation after adding 2.5 volumes (vol.) of absolute ethanol. The RNA was further purified by DNase I (Promega) treatment. RNA integrity was examined in a 2100 Bioanalyzer (Agilent Technologies). For the viroid library preparation, circular PSTVd molecules were purified from the pooled total RNA of each week by separating 10 µg of total RNA by 5% polyacrylamide-8 M urea denaturating gel electrophoresis (5 %-dPAGE). The circular PSTVd (cPSTVd) molecules were eluted from the gel and precipitated with 2.5 vol. of ethanol. In order to prepare the 2 complementary DNA (cDNA) libraries, 1 µg of purified cPSTVd RNA was separately subjected to reverse transcription (RT) using a primer binding at either position 280- to 269 (L1) or 10 to 354 (L2) of PSTVd-RG1, respectively, for each week. The resulting cDNA libraries were amplified by polymerase chain reaction (PCR) with the primers F1/R1 for L1 and F2/R2 for L2 followed by indexing of each library.
attributes.tag: GEO Accession
attributes.value: GSM4434546