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| Status |
Public on Apr 12, 2021 |
| Title |
CloneSeq: A Highly Sensitive Single-cell Analysis Platform for Comprehensive Characterization of Cells from 3D Culture |
| Organisms |
Homo sapiens; Mus musculus |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
Single-cell assays have revealed the scope and importance of heterogeneity in many biological systems. However, low sensitivity makes it difficult to delineate biological variation from noise, and as a result, the capacity of such assays to identify cellular state differences that arise from genes expressed at low-to-mid abundance is highly limited. To overcome this limitation, we developed CloneSeq, a microfluidics-based technology for 3D-culturing and RNA sequencing (RNA-seq) of small clones. CloneSeq shows that unlike random cell groups, clonal cancer cells originating from a single cell share similar transcriptional profiles. CloneSeq also dramatically improves sensitivity compared to standard single-cell RNA-seq. CloneSeq analysis of non-small cell adenocarcinoma cells revealed the presence of novel cancer-specific subpopulations, including cancer stem-like cells (CSLCs). Based on expression signatures, we identify CSLCs in highly proliferative clones, suggesting that our 3D culturing system promotes stemness. Supporting this, ESCs grown within the 3D hydrogel spheres retained their pluripotent state in the absence of pluripotent media. Moreover, reprogramming efficiency of mouse embryonic fibroblasts within the 3D spheres was significantly improved relative to that of cells grown on gelatin-coated plates. Our results highlight CloneSeq as an extremely useful tool for capturing lowly-expressed genes within clones, and for identifying rare cell populations such as stem-like and cancer-initiating cells.
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| Overall design |
Bulk RNA-seq of human (PC9) cells that were genomically barcoded and expanded inside hydrogel spheres (3D) and in a standard tissue culture (2D); and a bulk RNA-seq analysis of mouse embryonic stem (ES) cells grown in hydrogel spheres (3D) and on gelatin (2D) without supporting factors (2i/LIF) for 4 days, with 2 replicates for each experiment.
CloneSeq analysis of human (PC9) cells with two replicates; a single cell RNA-seq (inDrop) analysis of human (PC9); a single cell RNA-seq (inDrop) analysis of human (PC9) cells that were genomically barcoded and expanded inside hydrogel spheres (3D) with three replicates; and a bulk RNA-seq analysis of mouse embryonic stem (ES) cells grown in hydrogel spheres (3D) with doxycline, with and without supporting factors (2i/LIF) for 4 days, with 3 replicates for each experiment; and a CloneSeq and a scRNAseq anlysis of ES cells grown with dox.
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| Contributor(s) |
Bavli D, Sun X, Kozulin C, Ennis D, Motzik A, Ram O, Meshorer E, Buxboim A |
| Citation(s) |
34010629, 34632413 |
| Submission date |
Aug 07, 2020 |
| Last update date |
Oct 14, 2021 |
| Contact name |
Xue Sun |
| E-mail(s) |
xue.sun@mail.huji.ac.il
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| Organization name |
The Hebrew University of Jerusalem
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| Department |
Biochemistry department
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| Lab |
Oren Ram Lab
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| Street address |
Hebrew University, Givat Ram campus
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| City |
Jerusalem |
| ZIP/Postal code |
92000 |
| Country |
Israel |
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| Platforms (2) |
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| Samples (27)
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| Relations |
| BioProject |
PRJNA655931 |
| SRA |
SRP276692 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE155888_Barcode_1_list.txt.gz |
1.3 Kb |
(ftp)(http) |
TXT |
| GSE155888_Barcode_2_list.txt.gz |
1.3 Kb |
(ftp)(http) |
TXT |
| GSE155888_RAW.tar |
71.3 Mb |
(http)(custom) |
TAR (of BED, TXT) |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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