GSM7290617: Masopust_038_A [HTO]; Mus musculus; OTHER
Spleens were prepared through physical dissociation. Red blood cells in spleen samples were lysed using Ammonium-Potassium-Chloride lysis buffer (155mM ammonium, 1mM K+, 155mM Cl-, 0.1mM EDTA) lysis buffer, 2 minutes at room temperature. Mammary fat pad and EO771 tumors were digested in 1mg/mL collagenase IV (C6885, Sigma-Aldrich), 10% FBS, 0.5mM MgCl2, 0.5mM CaCl2, 1mM HEPES, 2μM L-glutamine, RPMI solution for 30 minutes in a shaking 37oC incubator prior to processing in gentleMACS C tubes and dissociator. MFP and tumor suspensions were subsequently separated on a 44:67 Percoll® (17-081-09, Cytiva) gradient. Suspensions were stained with Live/Dead dye (Ghost-Dye 780, Tonbo), fluorescent-conjugated antibodies (CD8a (53-6.7), CD44 (IM7), Thy1.1 (HIS51), CD45.1 (A20), Thy1.2 (30-H12)), and oligonucleotide-conjugated Biolegend TotalSeqTM A antibodies (1 hour, room temperature). P14 (Live, CD8a+, Thy1.1+), OT-I (Live, CD8a+, CD45.1+), and endogenous polyclonal CD8+ (Live, CD8a+, CD44+, Thy1.1-, CD45.1-) T cells were sorted using a FACS Aria II (BD Biosciences). The CITE-seq experiment was performed following “BioLegend's TotalSeqTM-A Antibodies and Cell Hashing with 10x Single Cell 3' Reagent Kit v3 or v3.1 (Single Index) Protocol”. Briefly, the cell suspension was loaded onto a 10X Genomics Chromium Controller at a concentration of 1,400 cells per microliter to target ~60,000 cells per two technical replicates. The single cells, reagents, and 10X Genomics gel beads were encapsulated into individual nanoliter-sized Gelbeads in Emulsion (GEMs) and then reverse transcription of poly-adenylated mRNA was performed inside each droplet. The 10X Genomics Chromium Single Cell 3′ v3.1 dual index protocol was used to prepare RNA. The Biolegend Protocol was used to prepare the hashtag-derived-tag (HDT) and antibody-derived-tag (ADT) libraries. 50,000 or 10,000 sequencing reads per cell for scRNA-seq or ADT&HDT libraries, respectively, were generated on the Illumina NovaSeq S4 flow cell.