GSM8829443: Multiplexed Cell lines 4T1WT, 66CL4WT, AT3, PY230; Mus musculus; RNA-Seq
Harvesting of confluent cells was performed through incubation with 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich) for 5 min and centrifugation at 400 × g for 5 min for subsequent counting with a Bürker chamber. A total of 2 x 106 cells from each mouse tumor cell line were incubated for 30 minutes at 4°C with a unique TotalSeq-A cell hashing antibody specific against mouse CD45 and MHC class I (1:250 diluted, HTO; TotalSeq-A0301 - TotalSeq-A0309 (BioLegend)). After incubation, cells were washed and centrifuged at 400 x g, 5 min, 4°C. The supernatant was discarded, the cell pellet was subsequently resuspended in 100 µl Cell Multiplexing Oligonucleotides (CMOs) and incubated at room temperature (RT) for 5 min (CMO01 – CMO09 (10X Genomics)). Samples were washed again (400 x g, 5 min, 4°C) and resuspended in PBS supplemented with 1% bovine serum albumin (BSA) for sorting. DAPI was added to the cells to sort out live cells, using a BD FACSAria II cell sorter (BD Biosciences). The tagged sorted cells were pooled (pool 1: D2A1 + MVT1 + EMT6 + MET1 + 4T1-hot; pool 2: 4T1-cold + 66CL4 + AT3 + PY230) and loaded on a Chromium GemCode Single Cell Instrument (10X Genomics) to generate single cell Gel beads-in-EMulsion (GEM). The single cell (sc)RNA-seq libraries were prepared using the GemCode Single Cell 3′ Gel Bead and Library kit, version Next GEM v3.1 according to the manufacturer's instructions (10X Genomics, User Guide CG000388) with the addition of the amplification primer 5'-GTGACTGGAGTTCAGACGTGTGCTCTTCCGAT*C*T-3´ (3 nM) during cDNA amplification to enrich the TotalSeq-A hashing protein oligos. Size selection with SPRIselect Reagent Kit (Beckman Coulter, B23318) was used to separate amplified cDNA molecules for 3′ gene expression, hashing protein and hashing lipid construction. TotalSeq-A protein library construction, including sample index PCR using Illumina's Truseq Small RNA primer sets, and SPRIselect size selection was performed according to the manufacturer's instructions. The cDNA content of pre-fragmentation and post-sample index PCR samples was analyzed using the 5200 Fragment Analyzer (Agilent). Sequencing libraries were loaded on an Illumina NovaSeq flow cell at VIB Nucleomics Core with sequencing settings according to the recommendations of 10X Genomics.