Single-cell RNA sequencing of tumor-infiltrating CD45+immune cells from E.G7 tumors with MDM2 knockdown
Single-cell RNA-Seq libraries were prepared using SeekOne DD Single Cell 3 library preparation kit (SeekGene Catalog No.K00202). Briefly, appropriate number of cells were mixed with reverse transcription reagent and then added to the sample well in SeekOne chip S3. Subsequently Barcoded Hydrogel Beads (BHBs) and partitioning oil were dispensed into corresponding wells separately in chip S3. After emulsion droplet generation reverse transcription were performed at 42for 90 minutes and inactivated at 85 for 5 minutes. Next, cDNA was purified from broken droplet and amplified in PCR reaction. The amplified cDNA product was then cleaned, fragmented, end repaired, A-tailed and ligated to sequencing adaptor. Finally, the indexed PCR were performed to amplified the DNA representing 3 polyA part of expressing genes which also contained Cell Barcode and Unique Molecular Index. The indexed sequencing libraries were cleanup with VAHTS DNA Clean Beads (Vazyme N411-01), analyzed by Qubit (Thermo Fisher Scientific Q33226) and Bio-Fragment Analyzer (Bioptic Qsep400). The libraries were then sequenced on illumina NovaSeq X Plus with PE150 read length.