GSM8860679: pWT_E185_lung_1; Mus musculus; RNA-Seq
RNA was extracted using TRIzol reagent (Invitrogen). 1 μg total RNA was used for following library preparation. RNA-Seq libraries were prepared with VAHTS mRNA-Seq V3 Library Prep Kit for Illumina (Vazyme, NR611). The poly(A) mRNA isolation was performed using Oligo(dT) beads. Using divalent cations and high temperatures, mRNA fragmentation was achieved and the priming process was carried out using random primers. Then first strand cDNA and the second-strand cDNA were synthesized. Once the double-stranded cDNA was purified, adaptors were added to both ends after end repair and dA-tailing. Size selection of Adaptor-ligated DNA was then performed using DNA Clean Beads. Each sample was then amplified by PCR using P5 and P7 primers and the PCR products were validated. Libraries were finally subjected to 150 bp paired-end sequencing using the Illumina NovaSeq 6000 instrument. For Smart-Seq2 library preparation, the cDNA was synthesized and amplified using the Single Cell Full-Length mRNA-Amplification Kit (Vazyme, N712). The full-length cDNA was assessed for quality and quantity using QIAxcel Advanced (QIAGEN) and Qubit 3.0 (ThermoFisher). Then RNA-Seq libraries were constructed from 1 ng cDNA using the TruePrep DNA Library Prep Kit V2 for Illumina (Vazyme, TD503). Amplified libraries were purified with VAHTS DNA Clean Beads (Vazyme, N411) and sequenced on the Illumina NovaSeq 6000.