title: GSM9012049: iMacs and MoMs - HTO; Homo sapiens; OTHER
library_construction_protocol: Cells from all conditions were generated, collected, processed and sequenced in parallel. On the day of isolation, cultures were enzymatically dissociated with 30 minutes of Accutase (ThermoFisher) followed by brief mechanical dissociation with a fire polished pipette. Single cell suspensions were transferred to phosphate buffered saline (PBS) containing 1:1000 eBioscience™ Fixable Viability Dye eFluor™ 780 (ThermoFisher) and incubated on ice for 20 minutes. Samples were maintained in dark conditions from this point onwards. Samples were centrifuged and washed in fluorescence-activated cell sorting (FACS) buffer containing PBS, 2% foetal calf-serum, 1% anti-anti and 0.5mM EDTA (ThermoFisher) before being resuspended in Human TrueStain FcX blocking buffer (Biolegend) diluted 1:10 in FACS buffer and incubated for 10 minutes on ice. Brilliant Violet 711 anti-human CD45 (1:100) and a unique TotalSeq-A hashtag oligo (HTO) (A-0251, A-0252, A-0253, A-0255, A-0256, A-0258, A-0256, A-0259, A-0260) were added directly to each tube and incubated for 30 minutes on ice. Samples were subsequently centrifuged, resuspended in FACS buffer and strained (100µm strainer) into FACS tubes. Cells were sorted into 1.5ml microcentrifuge tubes containing complete RPMI media Cells were loaded onto a Chromium Controller (10X Genomics) for single-cell partitioning, using Chromium Next GEM Chip G Single Cell Kit (10X Genomics), and single-cell 3' Reagent Kits v3.1 (10X Genomics).  A library consisting of iMACs and MoMs was sequenced on an Illumina Nextseq 500 at the Glasgow Polyomics facility.