Porcine high-resolution RNA isoform variation analysis by combining the advantages of single-cell RNA and nanopore sequencing. SusIA

The scRNA-seq library was generated utilising isolated cells and cell lysis from various tissues, including the spleen, liver, retina, peripheral blood mononuclear cells (PBMC), lung, brain, visceral adipose (Adipose-V), subcutaneous adipose (Adipose-S), and intestine. The remaining cDNA was stored in the -80°C fridge and then used for targeted capture and sequencing. The amplified and barcoded full-length cDNA derived from the previous procedure was subjected to the construction of the nanopore library. Initially, cDNA was amplified using adjusted bridge primers in combination with 2X LongAmp Taq HS Master Mix (M0533L, NEB). The PCR product was purified using 0.65x SPRIselection beads and then subjected to a second round of PCR with barcode primers from the SQK-PCB109 kit (nanopore. XXX PCR-cDNA barcoding kit) and SQK-PBK004 (low input PCR barcoding kit).