Mitochondria transfer from immune cells enables lymph node metastasis
Mitochondria transfer from immune cells enables lymph node metastasis
After tumor-immune co-cultures, cells were resuspended in FACS buffer (PBS in 0.5% BSA) and stained with DAPI and CD45. Live tumor cells (TdTomato+CD45-DAPI-) were sorted into Dendra2+ and Dendra2- subpopulations and pelleted immediately. Total RNA was isolated using Qiagen RNEasy Plus isolation kit. 1) mRNA enrichment and purification: Oligo dT Selection (mRNA enrichment): Use oligo dT beads to enrich mRNA with poly A tail. 2) RNA fragment and reverse transcription Fragment the RNA and first-strand cDNA was generated using random N6-primed reverse transcription, followed by a second-strand cDNA synthesis with dUTP instead of dTTP. 3) End repair, add A and adaptor ligation The synthesized cDNA was subjected to end-repair and then was 3' adenylated. Adaptors were ligated to the ends of these 3' adenylated cDNA fragments; 4) PCR amplification Prior to PCR amplification, the dUTP-marked strand is selectively degraded by Uracil-DNA-Glycosylase(UDG). The remaining strand is amplified to generate a cDNA library suitable for sequencing. Many rounds of PCR amplification were performed to enrich the purified cDNA template using PCR primer; 5) Single strand separation and cyclization Denature the PCR product by heat and the single strand DNA is cyclized by splint oligo and DNA ligase; 6) DNA nanoball synthesis