|
| Status |
Public on Sep 16, 2020 |
| Title |
HyPR-seq: Single-cell quantification of chosen RNAs via hybridization and sequencing of DNA probes (bulk RNA-seq dataset) |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by high throughput sequencing
|
| Summary |
Single-cell quantification of RNAs is important for understanding cellular heterogeneity and gene regulation, yet current approaches suffer from low sensitivity for individual transcripts, limiting their utility for many applications. Here we present HyPR-seq, a method to sensitively quantify the expression of 10 to 100 chosen genes in single cells. HyPR-seq involves hybridizing DNA probes to RNA, distributing cells into nanoliter droplets, amplifying the probes with PCR, and sequencing the amplicons to quantify the expression of chosen genes. HyPR-seq achieves high sensitivity for individual transcripts, detects non-polyadenylated and low-abundance transcripts, and can profile up to 100,000 single cells. We demonstrate how HyPR-seq can profile the effects of CRISPR perturbations in pooled screens, detect time-resolved changes in gene expression via measurements of gene introns, and detect rare transcripts and quantify cell type frequencies in tissue using low-abundance marker genes. By sensitively quantifying individual transcripts and directing sequencing power to genes of interest, HyPR-seq reduces costs by up to 100-fold compared to whole-transcriptome scRNA-seq approaches for these applications. HyPR-seq will be a powerful method for targeted RNA profiling in single cells.
|
| |
|
| Overall design |
We performed HyPR-seq in K562 cells (45 samples), THP1 cells (8 samples), and cells from wild-type and diabetic murine kidney (4 samples). HyPR-seq data from K562 cells includes 2 samples with probes targeting highly expressed genes and some introns, 1 sample representing a single-cell mixing experiment, and 42 samples representing a timecourse study to look at the kinetics of intron expression after promoter inhibition with CRISPR interference (3 samples each at 14 timepoints). THP1 samples include 4 replicates with LPS stimulation and 4 samples without. We also performed RNA sequencing on THP1 cells with and without LPS (3 replicates each) using the Smart-seq2 protocol in bulk. Kidney cells were obtained from 12-week old BTBR mice, 2 wt/wt and 2 ob/ob.
|
| |
|
| Contributor(s) |
Doughty BR, Engreitz JM, Chen F |
| Citation(s) |
33376219 |
| Submission date |
Sep 15, 2020 |
| Last update date |
Feb 02, 2021 |
| Contact name |
Jesse M Engreitz |
| E-mail(s) |
engreitz@stanford.edu
|
| Organization name |
Stanford University
|
| Street address |
Biomedical Innovation Building Rm 3700
|
| City |
Stanford |
| State/province |
CA |
| ZIP/Postal code |
94305 |
| Country |
USA |
| |
|
| Platforms (1) |
|
| Samples (6)
|
|
| This SubSeries is part of SuperSeries: |
| GSE158004 |
HyPR-seq: Single-cell quantification of chosen RNAs via hybridization and sequencing of DNA probes |
|
| Relations |
| BioProject |
PRJNA663610 |
| SRA |
SRP282486 |
Less...
More...