Multiplex single-cell profiling of chromatin accessibility by combinatorial cellular indexing. Simultaneous epitope and transcriptome measurement in single cells. Massively parallel single-nucleus RNA-seq with DroNc-seq. Massively parallel digital transcriptional profiling of single cells. Highly parallel genome-wide expression profiling of individual cells using nanoliter droplets. Droplet barcoding for single-cell transcriptomics applied to embryonic stem cells. Single-cell profiling of the developing mouse brain and spinal cord with split-pool barcoding. Comprehensive single-cell transcriptional profiling of a multicellular organism. Seq-Well: portable, low-cost RNA sequencing of single cells at high throughput. Mapping the mouse cell atlas by Microwell-Seq. Single-cell epigenomics: recording the past and predicting the future. Revealing the vectors of cellular identity with single-cell genomics. Scaling single-cell genomics from phenomenology to mechanism. scGESTALT provides a scalable platform to map lineage relationships between cell types in any system that permits genome editing during development, regeneration, or disease. Generating transgenic lines takes 6 months, and performing barcode editing and generating single-cell libraries involve 7 d of hands-on time. Here, we provide details for (i) generating transgenic zebrafish (ii) performing multi-timepoint barcode editing (iii) building scRNA-seq libraries from brain tissue and (iv) concurrently amplifying lineage barcodes from captured single cells. The recorded lineages are captured, along with thousands of cellular transcriptomes, to build lineage trees with hundreds of branches representing relationships among profiled cell types. The technique generates edits in the barcode array over multiple timepoints using Cas9 and pools of single-guide RNAs (sgRNAs) introduced during early and late zebrafish embryonic development, which distinguishes it from similar Cas9 lineage-tracing methods. We recently established a method, scGESTALT, that combines cumulative editing of a lineage barcode array by CRISPR–Cas9 with large-scale transcriptional profiling using droplet-based single-cell RNA sequencing (scRNA-seq). The phages were precipitated and purified by the double-polyethylene glycol (PEG) precipitation method described above.įull paper Login or join for free to view the full paper.Lineage relationships among the large number of heterogeneous cell types generated during development are difficult to reconstruct in a high-throughput manner. To produce chimeric phages, a colony containing the chimeric phage genome was used to inoculate a liquid culture and shaken overnight at 37 ☌. The inserted gene in the phage vector was amplified by PCR (forward primer: 5′- TTTGGAGCCTTTTTTTTGGAGATTTTCAAC-3′ reverse primer: 5′- CACCACCAGAGCCTGC-3′ PCR conditions: 95 ☌ for 3 min, then 11 cycles of 95 ☌ for 30 s, 52.3 ☌ for 30 s and 72 ☌ for 60 s) and Sanger sequenced (UC Berkeley core facility) to confirm the sequence of the chimeric phage genome. The recombinant plasmid was isolated using a QIAprep Spin Miniprep Kit. A single colony was selected and cultured in LB media with kanamycin (50 μg mL–1) and IPTG (0.1 M) in a shaking incubator at 37 ☌ overnight. The recombinant plasmid was then transformed into Mix and Go competent cells, which were plated on LB with kanamycin (50 μg mL–1) and IPTG (0.1 M) and incubated at 37 ☌ overnight. The g3p-N homologue was ligated into the M13-NotI-Kan vector using T4 DNA ligase. The desired products were isolated by gel electrophoresis and purified with QIAquick Gel Extraction Kit. The M13-NotI-Kan phage vector, in which a NotI restriction site was introduced between the N- and C-terminal domains of M13, was prepared previously.12 The extracted plasmid and M13-NotI-Kan vector were digested by KpnI-HF and NotI-HF. The cells were grown in LB media with ampicillin (10 μg mL–1), and the plasmid was isolated using the QIAprep Spin Miniprep Kit. A plasmid containing the g3p-N homologue of the source phage flanked by KpnI and NotI restriction sites (see the Supporting Information) was synthesized (IDT) and transformed into Mix and Go competent E. The RBP of M13 (g3p) was engineered to replace its N-terminal domain (g3p-N) with the homologous domain from a phage having specificity toward the target bacterial species (Table 1).
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