GSM9702026: A549 WT, untreated, barcode library transfected, mouse xenograft, 45 days, single-cell RNA-seq; Homo sapiens; RNA-Seq
For lineage tracing experiment in vitro, we trypsinized cells, resuspended them in DPBS for downstream analysis. For lineage tracing experiment in vivo, we used the Besttopcell mammalian cell tissues disassociation kit (BA3303) to get single-cell suspension of tumors, used MoFlo XDP (BeckMan Coulter) to sort GFP+ barcoded cancer cells and then performed the same protocols of Chromium. The cell suspension was passed through a 70 µm nylon cell strainer (ThermoFisher Scientific, 22-363-548) to remove clumps and debris, centrifuge the cells at 300 g for 5 min at 4 °C, discard the supernatant and the cells were re-suspended in PBS (Bioss, C7033) containing 5% FBS (Solarbio, s9020). Then cells were counted at count star. We observed that cell contained red blood cells in the count star instrument, next, we used red blood cell lysis buffer to remove red blood cell. Following a 15 min incubation at 4 °C, samples were centrifuged at 450 g for 5 min at 4 °C, discard the supernatant and wash twice times using PBS containing 5% FBS, Cells were then re-suspended in PBS containing 5%FBS. Next, cells were counted at count star and adjust the cell suspension concentration (600-1200 live cells per microliter). Then we followed similar protocols of the Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 (Dual Index) (10X Genomics) to finish single-cell RNA-seq. We followed protocols of the Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 (Dual Index) (10X Genomics) to finish single-cell RNA-seq. For lineage tracing experiment in vivo, we used excess full length cDNA sample from single-cell RNA-seq experiment workflow to improve the recovery of cellular barcodes. Specifically, we used a side PCR reaction, targeting the 3'UTR region between GFP and cellular barcodes. ~100 ng cDNA , side PCR primers (10 μM) and KAPA HiFi HotStart ReadyMix (KK2601) were mixed and we used the following protocols: 98 °C for 30 seconds, 24 cycles of 98 °C for 10 seconds and 65 °C for two minutes, followed by a final step at 65 °C for five minutes. Then the product was purified by 1.2x VAHTS® DNA Clean Beads.