title: ONT RNA-Seq of A549-Ctrl
design: Total RNA was extracted from the tissue using TRIzol reagent (Takara, Kyoto, Japan) according to the manufacturers instructions. RNA purity was tested using the Nano Photometer spectrophotometer (IMPLEN, Westlake Village, USA). cDNA libraries were constructed from 1 g of total RNA by using a cDNA-PCR Sequencing Kit (SQKPCS109) according to the protocol. Briefly, the reverse transcriptase was used to enrich full-length cDNAs and added defined PCR adapters to both ends of the first-strand of cDNA and following cDNA PCR for 14 circles with LongAmp Tag DNA polymerase (New England Biolabs, Ipswich, MA, USA) (8 min for elongation time). The PCR products were then subjected to ONT adaptor ligation using T4 DNA ligase (New England Biolabs, Ipswich, MA, USA). Agencourt XP beads (Beckman Coulter, Brea, USA) were used for DNA purification. The final cDNA libraries were subjected to FLOMIN109 flow cells and analyzed on a PromethION platform at Biomarker Technology Company (Beijing, China). Raw reads were first filtered with minimum average read quality score=7 and minimum read length=500bp. Ribosomal RNA were discarded after mapping to rRNA database. Next, full-length, non-chemiric (FLNC) transcripts were determined by searching for primer at both ends of reads. Clusters of FLNC transcripts were obtained after mapping to reference genome with mimimap2, and consensus isoforms were obtained after polishing within each cluster by pinfish.
library_name: AU437-A-ONT-1