GSM3832742: ccr7ko_tumor_adt; Mus musculus; OTHER
Prior to sorting, cells were stained with a mix of fluorescent antibodies and antibodies which had been conjugated to oligonucleotide barcodes using the Thunder-Link PLUS Oligo Conjugation kits (Expedeon) according to manufacturer's instructions. The sorted cells were encapsulated using the 10X Chromium platform, and libraries were prepared as previously described (Stoeckius, M. et al. Nat. Methods, doi:10.1038/nmeth.4380, 2017), with minor modifications. Briefly, cDNA amplification was performed in the presence of 2pM of an antibody-oligo specific primer to increase yield of antibody derived tags (ADTs). The amplified cDNA was then separated by SPRI size selection into cDNA fractions containing mRNA derived cDNA (>300bp) and ADT-derived cDNAs (<180bp), which were further purified by additional rounds of SPRI selection. Independent sequencing libraries were generated from the mRNA and ADT cDNA fractions, which were quantified, pooled and sequenced together on an Illumina Nextseq to a depth of 80 million reads per gene expression library and 20 million reads per ADT library. Single cell suspensions were obtained from lung and LN by digestion with collagenase IV 0.25mg/ml (Sigma) at 37C for 30min (lung) or 25min (LN) followed by passing through a 70um cell strainer and RBC lysis (RBC lysis buffer, BioLegend) for 2min at room temperature. For flow cytometry or FACS cells were stained in FACS buffer (PBS supplemented with 2% BSA and 5mM EDTA) with mAbs specific to CD45 (clone 30-F11, BioLegend), Siglec F (clone E50-2440, BD Pharmingen), CD11c (clone N418, Invitrogen), CD24 (clone M1/69, Invitrogen), CD103 (clone 2E7, BioLegend), I-A/I-E (clone M5/114.15.2, eBioscience), CD11b (clone M1/70, eBioscience)