BART-Seq: cost-effective massively parallelized targeted sequencing for genomics, transcriptomics, and single cell analysis [Pluripotency]

We describe a novel workflow named Barcode Assembly foR Targeted Sequencing, which is a highly sensitive, quantitative, and inexpensive technique for targeted sequencing of transcript cohorts (rBART-Seq) or genomic regions (gBART-Seq) from thousands of bulk samples or single cells in parallel. Multiplexing is based on a simple method that produces extensive matrices of diverse DNA barcodes attached to invariant primer sets, for generating amplicons with dual indices. Here, we used the rBART-Seq for RNA quantification, and for the analysis of developmental states of thousands of single human pluripotent stem cells maintained in different media (mTeSR™1, KSR-bFGF, and E8). Overall design: 11 selected transcripts and 4 external RNA spike-ins were co-amplified from single or multiple (2 to 32, incremented 2-fold) undifferentiated H9 human embryonic stem cells (hESCs) or BJ fibroblasts, as well as from four-fold dilution series (4-256 pg/well) of bulk RNA isolated from HMGU1 hiPSCs. The amplicons are barcoded using the rBART-seq method, and multiplexed for sequencing.