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

Summary

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.

Abstract

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.

Introduction

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 ....

Protocol

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 use¹³. Equilibrate with 95% O₂:5% CO₂ in an incubator.
      1. Prepare papain dissociation solution, 20 units/mL pa....

Results

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.......

Discussion

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 antibodies^7,19,20. However, this relies.......

Materials

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