Multi-step genomics on single cells and live cultures in sub-nanoliter capsules

Single-cell genomics encompasses a set of methods whereby hundreds to millions of cells are individually subjected to multiplexed assays including sequencing DNA, chromatin accessibility or modification, RNA, or combinations thereof 1,2. These methods enable unbiased, systematic discovery of cellular phenotypes and their dynamics 1–3. Many functional genomic methods, however, require multiple steps that cannot be easily scaled to high throughput, including assays on living cells. Here we develop capsules with amphiphilic gel envelopes (CAGEs), which selectively retain cells, mRNA, and gDNA, while allowing free diffusion of media, enzymes and reagents. CAGEs enable carrying out high-throughput assays that require multiple steps, including combining genomics with live-cell assays. We establish methods for barcoding CAGE DNA and RNA libraries, and apply them to measure persistence of gene expression programs by capturing the transcriptomes of tens of thousands of expanding clones in CAGEs. The compatibility of CAGEs with diverse enzymatic reactions will facilitate the expansion of the current repertoire of single-cell, high-throughput measurements and extend them to live-cell assays. RNA-seq data for the establishment of Capsule-based multi-step processing of single-cell in cultures. Data includes inC-RNAseq of fresh, frozen, and fixed cell lines, frozen PBMCs and cell lines cultured in CAGES. Washed cells were encapsulated into CAGEs, lyzed, washed, followed by mRNA capture via oligo-dT RT. cDNA was amplified in CAGEs, barcoded using split-pool implementation. Barcoded cDNA was used for 3’ end RNA-seq library preparation using NEBNext® Ultra™ II DNA Library Prep Kit for Illumina. Data also included single-cell inC-ATACseq data of cell lines, GFP-RFP gDNA amplicon sequencing of individual cells as well as plasmid barcoding test. Raw sequencing data are available via SRA. Fastq were demultiplexed to a sample resolution and contain all 4 reads. Read1 - transcript; read2 - PCR barcode, read3 - index; read - ligation barcode2, ligation barcode1, RT/ATAC/PCR barcode and UMI [for RNAseq]. Plasmid_sequencing: Sequencing data for the establishment of Capsule-based multi-step processing of single-cell and cultures. Data includes inC-seq barcoding of plasmid DNA processed in CAGES. DNA containing CAGEs were washed and plasmid DNA were amplified in CAGEs, barcoded using split-pool barcode addition. Barcoded DNA amplicon were indexed and sequenced using Illumina Raw sequencing data are available via SRA. Fastq were demultiplexed to a sample resolution and contain all 4 reads. Read1 - gDNA amplicon; read2 - PCR barcode, read3 - index; read - ligation barcode2, ligation barcode1 and CMV gDNA loci. Fresh_Fixed: RNA-seq data for the establishment of Capsule-based multi-step processing of single-cells and live cultures. Data includes inC-RNAseq of fresh and PFA fixed K562 cells processed in CAGES. Washed cells were encapsulated into CAGEs, lyzed (decrosslinked in PFA case), washed, followed by mRNA capture via oligo-dT RT. cDNA was amplified in CAGEs, barcoded using split-pool implementation. Barcoded cDNA was used for 3? end RNA-seq library preparation using NEBNext® Ultra? II DNA Library Prep Kit for Illumina. Raw sequencing data are available via SRA. Fastq were demultiplexed to a sample resolution and contain all 4 reads. Read1 - transcript; read2 - PCR barcode, read3 - index; read - ligation barcode2, ligation barcode1, RT/ATAC/PCR barcode and UMI [for RNAseq]. gDNA_Amplicons: Sequencing data for the establishment of Capsule-based multi-step processing of single-cell and cultures. Data includes inC-seq barcoding of GFP and RFP gDNA amplicon derived from a 1:1 mix of K562 and L1210 single-cells processed in CAGES. Washed cells were encapsulated into CAGEs, lyzed, washed, followed by mRNA capture via oligo-dT RT. gDNA targeted with CMV and RFP or GFP loci primers was amplified in CAGEs and barcoded using split-pool CAGE aproach. Barcoded gDNA amplicon were indexed and sequenced using Illumina Raw sequencing data are available via SRA. Fastq were demultiplexed to a sample resolution and contain all 4 reads. Read1 - gDNA amplicon; read2 - PCR barcode, read3 - index; read - ligation barcode2, ligation barcode1 and CMV gDNA loci. PBMCs: RNA-seq data for the establishment of Capsule-based multi-step processing of single-cells and live cultures. Data includes inC-RNAseq of cryopreserved PBMCs processed in CAGES. Thawed and washed cells were encapsulated into CAGEs, lyzed, washed, followed by mRNA capture via oligo-dT RT. cDNA was amplified in CAGEs and barcoded using inC-seq split-and-pool implementation. Barcoded cDNA was used for 3? end RNA-seq library preparation using NEBNext® Ultra? II DNA Library Prep Kit for Illumina. Raw sequencing data are available via SRA. Fastq were demultiplexed to a sample resolution and contain all 4 reads. Read1 - transcript; read2 - PCR barcode, read3 - index; read - ligation barcode2, ligation barcode1, RT and UMI. Cell_Culturing: RNA-seq data for the establishment of Capsule-based multi-step processing of single-cells and live cultures. Data includes inC-RNAseq of K562, L929 and induced pluripotent stems cells cultured in CAGES. After culturing, cells were lyzed followed by mRNA capture via oligo-dT RT. cDNA was purified, amplified and was used for 3? end RNA-seq library preparation using NEBNext® Ultra? II DNA Library Prep Kit for Illumina. Raw sequencing data are available via SRA. Fastq were demultiplexed to a sample resolution and contain all 4 reads. Read1 - transcript; read2 - PCR barcode, read3 - index; read4 - UMI [no barcodes were used] CAGE_Colonies: RNA-seq data for the establishment of Capsule-based multi-step processing of single-cells and live cultures. Data includes inC-RNAseq of mixed K562 and L1210 cells expanded into clones from single cells inside CAGEs in the presence of Decitabine, 5-Azacytidine or Vorinostat. Cells were pretreated with drugs and encapsulated into CAGEs, followed culturing. Cells were lysed after 0, 2, 4, 6 days followed by oligo dT mRNA capture. cDNA was barcoded using split-pool inC-seq approach and Barcoded DNA was used for scRNA-seq library construction Raw sequencing data are available via SRA. Fastq were demultiplexed to a sample resolution and contain all 4 reads. Read1 - transcript; read2 - PCR barcode, read3 - index; read - ligation barcode2, ligation barcode1, RT/ATAC/PCR barcode and UMI [for RNAseq].