In situ readout of DNA barcodes and single base edits facilitated by in vitro transcription

Molecular barcoding technologies that uniquely identify single cells are hampered by limitations in barcode measurement. Readout by sequencing does not preserve the spatial organization of cells in tissues, whereas imaging methods preserve spatial structure but are less sensitive to barcode sequence. Here we introduce a system for image-based readout of short (20-base-pair) DNA barcodes. In this system, called Zombie, phage RNA polymerases transcribe engineered barcodes in fixed cells. The resulting RNA is subsequently detected by fluorescent in situ hybridization. Using competing match and mismatch probes, Zombie can accurately discriminate single-nucleotide differences in the barcodes. This method allows in situ readout of dense combinatorial barcode libraries and single-base mutations produced by CRISPR base editors without requiring barcode expression in live cells. Zombie functions across diverse contexts, including cell culture, chick embryos and adult mouse brain tissue. The ability to sensitively read out compact and diverse DNA barcodes by imaging will facilitate a broad range of barcoding and genomic recording strategies

Data for "In situ readout of DNA barcodes and single base edits facilitated by in vitro transcription "

This dataset provides the raw and analyzed data as well as the code to recreate results reported in the paper "In situ readout of DNA barcodes and single base edits facilitated by in vitro transcription". The dataset is organized based on the figures and figure panels of the paper, with the codes provided as Matlab and R scripts. Please contact the corresponding author for any questions or comments regarding the data, the code, the methods, or the reagents used here.Related Publication:</p> In situ readout of DNA barcodes and single base edits facilitated by in vitro transcription</p> Askary, Amjad Caltech</p> Nature Biotechnology</p> https://doi.org/10.1038/s41587-019-0299-4</p>en

DS_TECH783497 – Supplemental material for Full Factorial Microfluidic Designs and Devices for Parallelizing Human Pluripotent Stem Cell Differentiation

<p>Supplemental material, DS_TECH783497 for Full Factorial Microfluidic Designs and Devices for Parallelizing Human Pluripotent Stem Cell Differentiation by Duncan M. Chadly, Andrew M. Oleksijew, Kyle S. Coots, Jose J. Fernandez, Shun Kobayashi, John A. Kessler and Akihiro J. Matsuoka in SLAS Technology</p

L'Athlète : journal hebdomadaire de tous les sports

03 août 19271927/08/03 (N533)-1927/08/03.Appartient à l’ensemble documentaire : Aquit

Developmental profiling of microRNAs in the human embryonic inner ear

<div><p>Due to the extreme inaccessibility of fetal human inner ear tissue, defining of the microRNAs (miRNAs) that regulate development of the inner ear has relied on animal tissue. In the present study, we performed the first miRNA sequencing of otic precursors in human specimens. Using HTG miRNA Whole Transcriptome assays, we examined miRNA expression in the cochleovestibular ganglion (CVG), neural crest (NC), and otic vesicle (OV) from paraffin embedded (FFPE) human specimens in the Carnegie developmental stages 13–15. We found that in human embryonic tissues, there are different patterns of miRNA expression in the CVG, NC and OV. In particular, members of the miR-183 family (miR-96, miR-182, and miR-183) are differentially expressed in the CVG compared to NC and OV at Carnegie developmental stage 13. We further identified transcription factors that are differentially targeted in the CVG compared to the other tissues from stages 13–15, and we performed gene set enrichment analyses to determine differentially regulated pathways that are relevant to CVG development in humans. These findings not only provide insight into the mechanisms governing the development of the human inner ear, but also identify potential signaling pathways for promoting regeneration of the spiral ganglion and other components of the inner ear.</p></div

Transcription factor targeting network plots, CVG vs NC comparison.

<p>Network plots showing differentially targeted transcription factors between CVG and NC tissues. Green nodes represent differentially expressed miRNAs, while orange and purple nodes represent significantly (false discovery adjusted <i>p</i> < 0.10) targeted transcription factors more targeted in either CVG (purple) or NC (orange) tissue. Transcription factor node size is proportional to the number of differentially expressed miRNAs targeting the gene.</p

Laser-microdissection of FFPE-human inner ear slides.

<p>(A) FFPE slides containing embryonic tissues. (B) Coronal section of a fetus with highlighted regions of interest (dashed black circles, CVG, NC, and OV). Further images are provided showing tissue before (C, E) and after (D, F) laser-captured microdissection of CVG (dashed white lines in D) and NC tissues (dashed white line in F). Other developmental regions also are highlighted. CVG: cochlear-vestibular ganglions, OV: otic vesicle, NC: neural crest; GG: geniculate ganglion, G-CVG: geniculate-cochleovestibular ganglions, E: epithelium; and NT: neural tube. Scale bar = 100 μm (B) and 150 μm (C-F).</p

REVIGO treemap of relevance similarity analysis on enriched pathways comparing CVG vs. NC (stage 13).

<p>REVIGO treemap summarizing Gene Ontology biological process categories over-represented in CVG cells compared to NC cells at stage 13. All terms are included with a FDR adjusted <i>p</i>-value cutoff at 0.05 from the enrichment analysis. The relevance similarity C-score (uniqueness) cut-off is chosen at 0.7. The size of each rectangle is proportional to the uniqueness for that category. Red circles indicate relevant CVG-development pathways.</p

Enriched gene sets, CVG vs NC comparison.

<p>Enriched gene sets, CVG vs NC comparison.</p

Differential expression of miRNA across tissues.

<p>Differentially expressed miRNA between three tissue types (CVG, NC, and OV) with <i>p</i> < 0.05 and false discovery adjusted <i>p</i> < 0.25, 0.50, or 0.10 (based on the smallest <i>p-</i>value out of the three possible tissue comparisons) are plotted for tissue from Carnegie stages 13, 14, and 15, respectively. MicroRNA from the miR-183 family is circled in red (stage 13) or green (stage 14).</p