GSM5811799: Sequential lineage tracing of HL60 cell differentiation with static barcode and scSLAM-seq, A13_MemorySeq384_3; Homo sapiens; OTHER
To facilitate lineage tracing in scSLAM-seq libraries, cellular barcodes (GBCs) were introduced using a lentiviral transduction strategy (Adamson et al., 2016). Given that the success of this experiment critically depended on the uniqueness of barcode sequence to each cell at the start of the experiment, i.e. low barcode collision rate, and the capture of clone cells (clones with the same barcodes) across different days, we used an experimental scheme in which the starting population of the HL60 cells were infected at a low (2%) multiplicity of infection (MOI). This scheme has two benefits: first, we obtained a small number of barcoded single cells (~2000 in 1 ml of media in each well of a 24-well plate) so that we could capture clone cells via plate-based SLAM-seq (scRNA-seq augmented by metabolic labeling) characterized of low throughput; second, co-culturing the small number of infected cells with a large population of uninfected cells enabled us to differentiate infected cells more conveniently, as a small number of cells are difficult to grow and differentiate. Single cells carrying barcodes and expressing the blue fluorescent protein (BFP) reporter were sorted (Sony SH800) at five timepoints, days 0, 1, 2, 3, and 5, during differentiation in the presence of ATRA. cDNA from single cells was prepared in a 96-well format as previously described (Tabula Muris Consortium, 2020) following alkylation and RNA cleanup (Erhard et al., 2019; Hendriks et al., 2019). Sequencing libraries were either reformatted into a 384-well format and prepared using TTP Mosquito automated liquid handlers, or in a 96- well format using a multichannel pipette. GBC sequencing libraries were prepared by dual PCR amplification to enrich for GBC cDNA and to add Illumina adapters and dual indexes complimentary to that cell's transcriptome sequencing library indexes. GBC sequencing libraries were spiked into transcriptome libraries at 1:10 and sequenced on the NextSeq or MiSeq platform (Illumina). Transcriptome libraries were sequenced separately using a NovaSeq5000 S2 300-cycle kit.