title: GSM8218322: LR25_MC38; Mus musculus; RNA-Seq
library_construction_protocol: At day 14 after B16F10 cells inoculation, or day 26 after MC38 cells inoculation, the tumors from 3 WT and 3 Lrrc25-/- mice were harvested, and 100 mm3 of each tumor was collected. Tumor tissues from 3 mice of the same group were randomly pooled into one mixed sample, and dissociated. Cell counts and viability were determined after removal of erythrocytes , and then debris and dead cells were removed using commercial kit. Fresh cells were washed twice in RPMI1640 and then resuspended at 1×106 cells per ml in 1×PBS containing 0.04% bovine serum albumin (BSA). Finally, CD45+ immune cells were isolated using magnetic beads and mixed with the original cell suspension at a ratio of 5:1. Single-cell RNA-Seq libraries were prepared using SeekOne® Digital Droplet 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. Subsequently Barcoded Hydrogel Beads (BHBs) and partitioning oil were dispensed into corresponding wells separately in chip. After emulsion droplet generation reverse transcription were performed at 42℃for 90 minutes and inactivated at 80℃ for 15 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 SPRI beads, quantified by quantitative PCR (KAPA Biosystems KK4824)