RNA-Seq of HRECs transduced with a FZD4-targeting shRNA lentivirus-replication 1
1. By utilizing the structural characteristic that most eukaryotic mRNAs carry a polyA tail, mRNAs were enriched using 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 were synthesized (for strand-specific libraries, dUTPs were used instead of dTTPs to incorporate dUTPs into the second-strand cDNAs. The high-fidelity DNA polymerase used in this method could not amplify uracil-containing DNA templates, thereby achieving 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 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, with the products subsequently solubilized in nuclease-free water. 7. After the initial library was constructed, a Qubit fluorescence quantier was used for concentration detection, followed by a Qsep400 high-throughput biofragment analyzer for fragment size detection.