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

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 to analyze cell subpopulations that emerge during 72 hours of Wnt/β-catenin pathway activation of H9 hESCs using recombinant Wnt protein (rWnt3a), a small molecule inhibitor of GSK-3 (CHIR99021), or Dox-inducible constitutively active β-catenin (ΔN90). 19 selected transcripts and 3 external RNA spike-ins were co-amplified from single cells isolated from differentiating H9 human embryonic stem cells (hESCs) at 0, 24 and 72 hours of Wnt pathway stimulation, as well as from bulk RNA derived from them (10/50 pg/well). The amplicons are barcoded using the rBART-seq method, and multiplexed for sequencing.