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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Neuroscience

Multiplexed Analysis of Retinal Gene Expression and Chromatin Accessibility Using scRNA-Seq and scATAC-Seq

Published: March 12th, 2021

DOI:

10.3791/62239

1Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, 2Department of Psychiatry and Behavioral Science, The Johns Hopkins Hospital, 3Department of Ophthalmology, The Johns Hopkins Hospital, 4Department of Neurology, The Johns Hopkins Hospital, 5Institute for Cell Engineering, Johns Hopkins University School of Medicine, 6Kavli Neuroscience Discovery Institute, Johns Hopkins University School of Medicine
* These authors contributed equally

Here, the authors showcase the utility of MULTI-seq for phenotyping and subsequent paired scRNA-seq and scATAC-seq in characterizing the transcriptomic and chromatin accessibility profiles in retina.

Powerful next generation sequencing techniques offer robust and comprehensive analysis to investigate how retinal gene regulatory networks function during development and in disease states. Single-cell RNA sequencing allows us to comprehensively profile gene expression changes observed in retinal development and disease at a cellular level, while single-cell ATAC-Seq allows analysis of chromatin accessibility and transcription factor binding to be profiled at similar resolution. Here the use of these techniques in the developing retina is described, and MULTI-Seq is demonstrated, where individual samples are labeled with a modified oligonucleotide-lipid complex, enabling researchers to both increase the scope of individual experiments and substantially reduce costs.

Understanding how genes can influence cell fate plays a key role in interrogating processes such as disease and embryonic progression. The complex relationships between transcription factors and their target genes can be grouped in gene regulatory networks. Mounting evidence places these gene regulatory networks at the center of both disease and development across evolutionary lineages1. While previous techniques such as qRT-PCR focused on a single gene or set of genes, the application of high-throughput sequencing technology allows for the profiling of complete cellular transcriptomes.

RNA-seq offers a glimpse into ....

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The use of animals for these studies was conducted using protocols approved by the Johns Hopkins Animal Care and Use Committee, in compliance with ARRIVE guidelines, and were performed in accordance with relevant guidelines and regulations.

1. MULTI-seq

  1. Media preparation
    1. Prepare and equilibrate ovomucoid inhibitor, 10 mg of ovomucoid inhibitor and 10 mg of albumin per mL of Earle's Balanced Salt Solution (EBSS), for 30 min prior to use

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This workflow lays out a strategy for investigation of developmental phenotypes and regulatory processes using single cell sequencing. MULTI-seq sample multiplexing enables an initial low-cost phenotyping assay while paired collection and fixation of samples for scRNA-seq and scATAC-seq allows for more in-depth investigation (Figure 1).

MULTI-seq barcoding enables the combined sequencing of multiple samples and their subsequent computational deconvolution. The sam.......

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The power of MULTI-seq stems from seamless integration of data from multiple experimental conditions or models and the enormous benefit in terms of cost and limiting batch effects. Utilizing MULTI-seq offers a laboratory unprecedented phenotyping depth. Non-genetic multiplexing methods such as cell hashing or nuclei hashing opened the door to multiplexed samples through the use of barcoded antibodies7,19,20. However, this relies.......

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We thank Linda Orzolek from the Johns Hopkins Transcriptomics and Deep Sequencing Core for help in sequencing the produced libraries and Lizhi Jiang for performing the ex vivo retinal explants.

....

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Name Company Catalog Number Comments
10 µL, 200 µL, 1000 µL pipette filter tips
10% Tween 20 Bio-Rad 1662404
100 µM Barcode Solution Request from Gartner lab https://docs.google.com/forms/d/1bAzXFEvDEJse_cMvSUe_yDaP
rJpAau4IPx8m5pauj3w/viewform?ts=5c47a897&edit_requested
=true
100% Ethanol Millipore Sigma E7023-500ML
100% Methanol Millipore Sigma 322415-100ML
10x Chip Holder 10x Genomics 1000195
10x Chromium controller & Accessory Kit 10x Genomics PN-120263
15mL Centrifuge Tube Quality Biological P886-229411
40 µm FlowMi Cell Strainer Bel-Art H13680-0040
50 µM Anchor Solution Sigma or request from Gartner lab https://docs.google.com/forms/d/1bAzXFEvDEJse_cMvSUe_yDaP
rJpAau4IPx8m5pauj3w/viewform?ts=5c47a897&edit_requested
=true
50 µM Co-Anchor Solution Sigma or request from Gartner lab https://docs.google.com/forms/d/1bAzXFEvDEJse_cMvSUe_yDaP
rJpAau4IPx8m5pauj3w/viewform?ts=5c47a897&edit_requested
=true
5200 Fragment Analyzer system Agilent M5310AA
70 um FlowMi cell strainer Bel-Art H13680-0070
Allegra X-12R Centrifuge VWR BK392302
Bovine Serum Albumin Sigma-Aldrich A9647
Chromium Next GEM Chip G 10x Genomics PN-1000120
Chromium Next GEM Chip H 10x Genomics PN-1000161
Chromium Next Gem Single Cell ATAC Reagent Kit v1.1 10x Genomics PN-1000175
Chromium Single Cell 3' GEM, Library & Gel Bead Kit v3.1 10x Genomics PN-1000121
Digitonin Fisher Scientific BN2006
Dissection microscope Leica
DNA LoBind Tubes, 1.5 mL Eppendorf 22431021
Dry Ice
EVA Foam Ice Pan Tequipment 04393-54
FA 12-Capillary Array Short, 33 cm Agilent A2300-1250-3355
Fisherbrand Isotemp Water Bath Fisher Scientific 15-460-20Q
Forma CO2 Water Jacketed Incubator ThermoFisher Scientific 3110
Glycerol 50% Aqueous solution Ricca Chemical Company 3290-32
Hausser Scientific Bright-Line Counting Chamber Fisher Scientific 02-671-51B
Illumina NextSeq or NovaSeq Illumina
Kapa Hifi Hotstart ReadyMix HiFi 7958927001
Low TE Buffer Quality Biological 351-324-721
Magnesium Chloride Solution 1 M Sigma-Aldrich M1028
Magnetic Separator Rack for 1.5 mL tubes Millipore Sigma 20-400
Magnetic Separator Rack for 200 µL tubes 10x Genomics NC1469069
MULTI-seq Primer Sigma or IDT See sequence list
MyFuge Mini Centrifuge Benchmark Scientific C1008
Nonidet P40 Substitute Sigma-Aldrich 74385
Nuclease-free water Fisher Scientific AM9937
P2, P10, P20, P200, P1000 micropipettes Eppendorf
Papain Dissociation System Worthington Biochemical Corporation LK003150
PBS pH 7.4 (1X) Fisher Scientific 10010-023
Qiagen Buffer EB Qiagen 19086
Refridgerated Centrifuge 5424 R Eppendorf 2231000655
RNase-free Disposable Pellet Pestles Fisher Scientific 12-141-368
RNasin Plus RNase Inhibitor Promega N2615
RPI primer Sigma or IDT See sequence list
Single Index Kit N, Set A 10x Genomics PN-1000212
Single Index Kit T Set A 10x Genomics PN-1000213
Sodium Chloride Solution 5 M Sigma-Aldrich 59222C
SPRIselect Reagent Kit Beckman Coulter B23318
Standard Disposable Transfer Pipettes Fisher Scientific 13-711-7M
TempAssure PCR 8-tube strip USA Scientific 1402-4700
Trizma Hydrochloride Solution, pH 7.4 Sigma-Aldrich T2194
Trypan Blue Solution, 0.4% (w/v) Corning 25-900-CI
Universal I5 primer Sigma or IDT See sequence list
Veriti Thermal Cycler Applied Biosystems 4375786
Vortex Mixer VWR 10153-838

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