GSM8267098: NK_Tissues_Tumor_Fasting; Mus musculus; RNA-Seq
Bone marrow was flushed using PBS + 2% FCS, then subjcted to red blood cell lysis before sorting. Spleen tissue was passed through 100uM strainers in PBS + 2% FCS, then subjcted to red blood cell lysis before sorting.Tumor tissues were digested in DNase I  and Collagenase 4 at 37C for 30 minutes then passed through a 100uM strainer before sorting. Cell sorting was performed on Aria II cytometers (BDBiosciences). NK cells were defined as Lineage- NK1.1+ CD49b+. Upon cell sorting and HTO staining, NK cells from each condition were mixed at equal ratios, washed once with PBS containing 1% BSA to a final concentration of 700-1,300 cells per ul. 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 PCR amplification per manual instruction. Around 15,000 cells were targeted. Samples were multiplexed together on one lane of 10X Chromium (using Hash Tag Oligonucleotides - HTO) following previously published protocol79. Final libraries were sequenced on Illumina NovaSeq S4 platform (R1 – 28 cycles, i7 – 8 cycles, R2 – 90 cycles). The cell-gene count matrix was constructed using seqc (https://github.com/dpeerlab/seqc).