Probe-Seq enables transcriptional profiling of specific cell types from heterogeneous tissue by RNA-based isolation
- Howard Hughes Medical Institute, Harvard Medical School, United States
Figures
Figure 1 with 5 supplements
Isolation and transcriptional profiling of specific BC subtypes from the adult mouse retina.
(a) Schematic of Probe-Seq for the adult mouse retina. The retina was dissociated into single cells, fixed, and permeabilized. Cells were incubated with gene-specific probe sets against Vsx2 and Grik…
Figure 1—figure supplement 1
Quality control of mouse Probe-Seq RNA sequencing.
(a) Log2-transformed read distribution plot for sequenced mouse Vsx2/Grik1 Probe-Seq samples. (b) Dendrogram of read counts shows clustering of Vsx2-, Grik1-, and Grik1+ samples. (c) Plots of …
Figure 1—figure supplement 2
BC markers are enriched in the Vsx2+/Grik1+ and Vsx2+/Grik1- populations.
A heatmap representing relative expression levels of major retinal cell class markers previously identified by scRNA sequencing (Macosko et al., 2015) that are differentially expressed (adjusted …
Figure 1—figure supplement 3
Validation of a Grik1+ population-enriched transcript, Tpbgl, by SABER-FISH.
(a) Heapmaps of unbiased top 20 enriched genes for each population (Vsx2-, left; Grik1-, middle; Grik1+, right). (b) Quantification of Tpbgl transcripts in Vsx2-, Grik1-, Grik1+ populations based on …
Figure 1—figure supplement 4
Gene body coverage of Live and Probe-Seq cells show 3’ bias in the Probe-Seq population.
Graphs showing gene body coverage of mapped reads from Live (left panel) and Probe-Seq (right panel) cells. Due to cDNA generation using Oligo(dT), cDNA from degraded RNA is biased towards the 3’ …
Figure 1—figure supplement 5
Probe-Seq stain index is correlated with the number of oligos and the expression level.
(a) Quantification of number of puncta per positive cell for Grik1, Grm6, and Neto1 in the mouse retina. (b) Schematic of the experiment. For each gene, 48, 24, or 12 randomly-chosen oligos were …
Figure 2 with 2 supplements
Transcriptional profiling of nuclear RNA isolated from specific BC subtypes from frozen human retina.
(a) Schematic of Probe-Seq for the fresh frozen adult human retina. Single nuclei were prepared and then fixed. Nuclei were incubated with a gene-specific probe set for GRM6 and then incubated with …
Figure 2—figure supplement 1
Isolation and transcriptional profiling of specific BC subtypes from the frozen mouse retina.
(a) Schematic of Probe-Seq for the fresh frozen adult mouse retina. Single nuclei were isolated and fixed. Nuclei were incubated with a gene-specific probe set for Grik1 and then incubated with …
Figure 2—figure supplement 2
Quality control of human nuclear Probe-Seq RNA sequencing.
(a) Log2-transformed read distribution plot for sequenced human GRM6 Probe-Seq samples. (b) Dendrogram of read counts shows clustering of GRM6- and GRM6+ samples. (c) Plot of frequencies of p-values …
Figure 3 with 2 supplements
Isolation and transcriptional profiling of ISC/EBs from the adult Drosophila midgut.
(a) Schematic of Probe-Seq for the adult Drosophila midgut. Midguts from 7 to 10 day old female flies were dissociated into single cells and fixed. Cells were incubated with a gene-specific probe …
Figure 3—figure supplement 1
Quality control of Drosophila Probe-Seq RNA sequencing.
(a) Log2-transformed read distribution plot for sequenced Drosophila esg Probe-Seq samples. (b) Dendrogram of read counts shows clustering of esg- and esg+ samples. (c) Plot of frequencies of p-value…
Figure 3—figure supplement 2
Heatmap of ISC/EB and EC markers based on DamID transcriptional profiling.
A heatmap representing relative expression levels of Drosophila ISC/EB and EC markers previously identified by DamID transcriptional profiling (adjusted p-value<0.05) between esg-, and esg+ …
Figure 4 with 3 supplements
Probe-Seq identifies the transcriptional landscape of chick central retinal progenitor cells that give rise to the high acuity area.
(a) Schematic of Probe-Seq for the developing HH28 chick retina. The chick retina was dissociated into single cells and fixed. Cells were incubated with a gene-specific probe set for FGF8 and …
Figure 4—figure supplement 1
Uniform distribution of mapped reads from embryonic chick Probe-Seq for FGF8.
A representative graph of gene body coverage of mapped reads from chick Probe-Seq.
Figure 4—figure supplement 2
Quality control of developing chick retina Probe-Seq RNA sequencing.
(a) Log2-transformed read distribution plot for sequenced chick Probe-Seq samples. (b) Dendrogram of read counts shows clustering of FGF8- and FGF8+ samples except FGF8+−1. (c) Plot of frequencies …
Figure 4—figure supplement 3
Early differentiation markers are enriched in the FGF8- population.
A heatmap of top 50 unbiased differentially expressed genes between FGF8+ and FGF8- populations in the developing chick retina.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Mus musculus) | Vsx2 | Ensembl: ENSMUSG00000021239 | ||
| Gene (Mus musculus) | Grik1 | Ensembl: ENSMUSG00000022935 | ||
| Gene (Mus musculus) | Tpbgl | Ensembl: ENSMUSG00000096606 | ||
| Gene (Mus musculus) | Grm6 | Ensembl: ENSMUSG00000000617 | ||
| Gene (Mus musculus) | Neto1 | Ensembl: ENSMUSG00000050321 | ||
| Gene (Mus musculus) | Gad1 | Ensembl: ENSMUSG00000070880 | ||
| Gene (Homo sapiens) | GRM6 | Ensembl: ENSG00000113262 | ||
| Gene (Homo sapiens) | CDH12 | Ensembl: ENSG00000154162 | ||
| Gene (Drosophila melanogaster) | esg | FlyBaseID: FBgn0001981 | ||
| Gene (Drosophila melanogaster) | Sox100B | FlyBaseID: FBgn0024288 | ||
| Gene (Gallus gallus) | FGF8 | Ensembl: ENSGALG00000007706 | ||
| Gene (Gallus gallus) | BMP2 | Ensembl: ENSGALG00000029301 | ||
| Strain, strain background (Mus musculus) | CD1 | Charles River Laboratories | 022 | |
| Strain, strain background (Drosophila melanogaster) | Oregon-R | |||
| Strain, strain background (Gallus gallus) | White Leghorn | Charles River Laboratories | ||
| Sequence-based reagent | Variable oligos | This Paper | See Supplementary Table for sequences | |
| Commercial assay or kit | RecoverAll Total Nuclear Isolation Kit | Thermo Fisher Scientific | AM1975 | |
| Commercial assay or kit | SMART-Seq v.4 Ultra Low Input RNA kit | Takara Bio | 634890 | |
| Commercial assay or kit | Nextera XT DNA Library Prep Kit | Illumina | FC1311096 |
Additional files
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Source code 1
R code for differential expression alaysis.
- https://cdn.elifesciences.org/articles/51452/elife-51452-code1-v1.zip
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Supplementary file 1
List of oligos used to generate gene-specific probe sets, fluorescent oligos, and hairpins.
- https://cdn.elifesciences.org/articles/51452/elife-51452-supp1-v1.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/51452/elife-51452-transrepform-v1.docx
