GSM8864603: WT-1; Nicotiana tabacum; RNA-Seq
mrna extraction by Trizol 1) By utilizing the structural characteristic that most eukaryotic mRNAs carry a polyA tal, mRNAs with polyA tails were enriched by Oligo(dT) magnetic beads; 2) The purified mRNAs were cleaved into small fragments with fragmentation buffer at a suitable temperature;3) First-strand cDNAs were produced by reverse transcription using a random hexamer primer;4) Second-strand cDNAs are synthesized (strand-specific lilbrary: dUTPs were used instead of dTTPs in thesecond-strand synthesis to incorporate dUTPs in the second-strand cDNAs, while the high-fidelity DNApolymerase used in this method could not amplify uracil-containing DNA templates, thus realizing thestrand-specificity), while simultaneously performing end repair and dA-Tailing;5) Sequencing adapter ligation was performed, followed by DNA magnetic bead purification and fragment selectionafter ligation was completed to yield a library with 250-350 bp insert fragments;6) The ligated products were amplified by PCR and purified again using DNA magnetic beads, where the productswere solubilized with nuclear-free water:7) After the initial library was constructed, a Qubit fluorescence quantifier was used for concentration detectionfollowed by a Qsep400 high-throughput biofragment analyzer for fragment size detection
GEO
GSM8864603_r1