library_construction_protocol: After tumor harvested, tissues were washed in ice-cold RPMI1640 and dissociated using Multi-tissue dissociation kit 2 (Miltenyi) from Miltenyi Biotec as instructions. Cell count and viability was estimated using fluorescence Cell Analyzer (Countstar® Rigel S2) with AO/PI reagent after removal erythrocytes (Miltenyi 130-094-183) and then debris and dead cells removal was decided to be performed or not (Miltenyi 130-109-398/130-090-101). Finally, fresh cells were washed twice in the RPMI1640 and then resuspended at 1×106 cells per ml in 1×PBS and 0.04% bovine serum albuminat. Single-cell RNA-Seq libraries were prepared using SeekOne® MM Single Cell 3' library preparation kit (SeekGene Catalog No.). Briefly, appropriate number of cells were loaded into the flow channel of SeekOne® MM chip which had 170,000 microwells and allowed to settle in microwells by gravity. After removing the unsettled cells, sufficient Cell Barcoded Magnetic Beads (CBBs) were pipetted into flow channel and also allowed to settle in microwells with the help of a magnetic field. Next excess CBBs were rinsed out and cells in MM chip were lysed to release RNA which was captured by the CBB in the same microwell. Then all CBBs were collected and reverse transcription were performed at 37℃for 30 minutes to lable cDNA with cell barcode on the beads. Further Exonuclease I treatment were performed to remove unused primer on CBBs. Subsequently, barcoded cDNA on the CBBs was hybridized with random primer which had reads 2 SeqPrimer sequence on the 5' end and could extend to form the second strand DNA with cell barcode on the 3' end. The resulting second strand DNA were denatured off the CBBs, purified and amplified in PCR reaction. The amplified cDNA product was then cleaned to remove unwanted fragment and added to full length sequencing adapter and sample index by indexed PCR. The indexed sequencing libraries were cleanup with SPRI beads, quantified by quantitative PCR (KAPA Biosystems KK4824) and then sequenced on illumina NovaSeq 6000 with PE150 read length or DNBSEQ-T7 platform with PE100 read length.