Split-pool combinatorial barcoding sequencing of murine immune cells
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PROVIDER: PRJNA1065472 | ENA |
REPOSITORIES: ENA
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Project description:To establish an ultra-high-throughput single cell chromatin accessibility profiling method that is cost-effective and widely accessible, we built on sci-ATAC-seq (Cusanovich, D. A. et al. Science. 2015; Amini, S. et al. Nature Genetics. 2014) and SPLIT-seq (Rosenberg, A. B. et al. Science. 2018) to design SPATAC-seq, which in situ label chromatin fragment in the same single cell through combinatorial barcoding. Briefly, in SPATAC-seq, (1) fixed nuclei are transposed in 48 different wells by 48 unique Tn5 transposase, which containing barcoded adaptors and 5'-phosphorylation; (2) the nuclei from all wells are collected and redistributed into second and third 48-well plate in turn, where the next two rounds of indexing are achieved through into either end of the custom transposome, which result in the generation of more than 110,000 (48^3) unique barcode combinations. (3) the nuclei are pooled, split into sublibraries and lysed, and the DNA was amplified by polymerase chain reaction (PCR), which introduce illumina sequencing barcodes and complete libraries construction. (4) After sequencing, fastq files were demultiplexed according to the same four-barcode combinations. For profiling more cells in one sublibrary, we can increase the number of barcode combinations by increase the number of indexing of each round to 96, which can produce about 1 million combinations. To assess the fidelity of SPATAC-seq, we performed a species-mixing experiment with cultured human (K562) and mouse (Hepa) cells. Here, we tagged mixed permeabilized nuclei with only 8 barcoded transposome. In round 4, we generated eight sublibraries with different cell-recovery targets, which can be used to evaluating the stability of this method and the correlation between real doublet rates and theoretical value.
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