title: GSM9211940: IL10R treated B16, mRNA; Mus musculus; RNA-Seq
library_name: GSM9211940
library_construction_protocol: mice were treated as indicated and twelve hours after treatment mice were euthanized and tumors were harvested. Samples were then passed through a 100 μm filter and resuspended in 3 ml of 40% Percoll (Fisher Scientific). Samples were then centrifuged at 1500 rpm for 5 minutes. To remove red blood cells, Samples were resuspended in ACK Lysing Buffer (Thermo Fisher) on ice. Cells were then resuspended in FACS Buffer at 1-2 x 106 cells per 100 μL. All wells were treated with DAPI viability dye, washed and then incubated with Fc block. Cells were subsequently pooled according to treatment group. Samples were then sequenced and analyzed. The single-cell RNA-Seq of FACS-sorted cell suspensions was performed on Chromium instrument (10X Genomics) following the user guide manual for 3′ v3. FACS-sorted cells were washed once with PBS containing 1% bovine serum albumin (BSA) and resuspended in PBS containing 1% BSA. The viability of cells was above 80%, as confirmed with 0.2% (w/v) Trypan Blue staining (Countess II). Cells were captured in droplets. Following reverse transcription and cell barcoding in droplets, emulsions were broken and cDNA purified using Dynabeads MyOne SILANE followed by polymerase chain reaction (PCR) amplification per manual instruction. Between 5,000 to 20,000 cells were targeted for each sample. Samples were multiplexed together on one lane of 10X Chromium. Final libraries were sequenced on Illumina NovaSeq S4 platform (R1 – 28 cycles, i7 – 8 cycles, R2 – 90 cycles).