GSM9543858: MATEX_1_MC38_D14_WT_CD45pos_CD64neg; Mus musculus; RNA-Seq
MC38 tumors from CD64DTR and CD64WT were harvested at day 14 d.p.i., digested in Liberase TL 0,25mg/ml (Roche, #5401020001) DNAse 0,5mg/ml (Sigma, DN25-100MG) in CO2 independent medium (Gibco, 18045088) using GentleMacs Octo dissociator pre-set program (37C_m_TDK1, 41min at 37° under agitation). Cells were filtered on a 100 µm cell strainer and red blood cells were lysed using red blood cells lysis buffer 1X (Biolegend, 420301) diluted in water. After centrifugation (5min, 1500rpm), cells were incubated 10 min with Fcblock solution diluted in FACS buffer (BD, 553142, 1:100) and stained either with (1) DAPI (Invitrogen, 11534886, 1:10000) for the first experiment (batch #1), or (2) with DAPI, anti-CD45 APC-Cy7 (BD, 557659, 1:200) and anti-CD64 APC (Biolegend,139306, 1:200) (for the second experiment (batch #2) diluted in FACS buffer (PBS 1X, 0,5%BSA, 2mM EDTA). For batch#1, DAPI- cells were sorted while for batch#2, DAPI-CD45-, DAPI-CD45+CD64+, DAPI- CD45+CD64- populations were sorted separately. For batch #1, 15 000 cells form CD64WT and CD64DTR tumors were loaded. For Batch #2, 10000 cells form CD64WT and CD64DTR tumors were loaded. Library was performed following 10x Genomics instructions (Single Cell 3' V3.1 Next-GEM kit protocol).