GSM8578226: NS1; Mus musculus; RNA-Seq
RNA was extracted by Trizol method. 1) By utilizing the structural characteristic that most eukaryotic mRNAs carry a polyA tail, 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 library: dUTPs were used instead of dTTPs in the second-strand synthesis to incorporate dUTPs in the second-strand cDNAs, while the high-fidelity DNA polymerase used in this method could not amplify uracil-containing DNA templates, thus realizing the strand specificity), while simultaneously performing end repair and dA-Tailing; 5) Sequencing adapter ligation was performed, followed by DNA magnetic bead purification and fragment selection after 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 products were solubilized with nuclear-free water; 7) After the initial library was constructed, a Qubit fluorescence quantifier was used for concentration detection, followed by a Qsep400 high-throughput biofragment analyzer for fragment size detection; 8) The double-stranded DNA library obtained by PCR was subjected to single-stranded cyclization to obtain a single-stranded circular DNA library; 9) The final library was amplified with phi29 before uploading to make DNA nanoballs (DNB) with more than 300 copies of a molecule, and the DNBs were loaded into the sequencing chip and sequenced on the BGI sequencing platform.