Aug 22, 2025
scATAC-seq on mEBs
This protocol is a draft, published without a DOI.
- Jean-Benoît alanne1
- 1Department of Genome Sciences, University of Washington, Seattle, WA, USA
Protocol Citation: Jean-Benoît alanne 2025. scATAC-seq on mEBs. protocols.io https://protocols.io/view/scatac-seq-on-mebs-g76qbzrdx
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Full methods can be found in the Supplementary Information from Multiplex profiling of developmental cis-regulatory elements with quantitative single-cell expression reporters
Created: August 22, 2025
Last Modified: August 22, 2025
Protocol Integer ID: 225200
Keywords: function multicellular expression phenotypes from cre variant, cell quantitative expression reporter, multicellular expression phenotype, rna barcode stabilization via circularization, accessible chromatin, specific developmental cre, perturbed transcription factor, regions of accessible chromatin, transcription factor, cell reporter assay, quantitative expression reporter, dual rna cassette, multicellular, measurement of reporter expression, rna, bottleneck in genomic, genomic, cre variant, regulatory element, quantification tasks inherent to multiplex, developmental ci, chimeric cre, activity of developmental ci, synthetic cres at scale, cre
Funders Acknowledgements:
NHGRI
Grant ID: UM1HG011966
NHGRI
Grant ID: R01HG010632
Damon Runyon Cancer Research Foundation
Grant ID: DRG-2435-21
NHGRI
Grant ID: F31HG011576
National Heart, Lung, and Blood Institute
Grant ID: T32HL007828
NHGRI
Grant ID: F32HG011817
Abstract
The inability to scalably and precisely measure the activity of developmental cis-regulatory elements (CREs) in multicellular systems is a bottleneck in genomics. Here we develop a dual RNA cassette that decouples the detection and quantification tasks inherent to multiplex single-cell reporter assays. The resulting measurement of reporter expression is accurate over multiple orders of magnitude, with a precision approaching the limit set by Poisson counting noise. Together with RNA barcode stabilization via circularization, these scalable single-cell quantitative expression reporters provide high-contrast readouts, analogous to classic in situ assays but entirely from sequencing. Screening >200 regions of accessible chromatin in a multicellular in vitro model of early mammalian development, we identify 13 (8 previously uncharacterized) autonomous and cell-type-specific developmental CREs. We further demonstrate that chimeric CRE pairs generate cognate two-cell-type activity profiles and assess gain- and loss-of-function multicellular expression phenotypes from CRE variants with perturbed transcription factor binding sites. Single-cell quantitative expression reporters can be applied in developmental and multicellular systems to quantitatively characterize native, perturbed and synthetic CREs at scale, with high sensitivity and at single-cell resolution.
Troubleshooting
scATAC-seq on mEBs from the methods in: Multiplex profiling of developmental cis-regulatory elements with quantitative single-cell expression reporters
Single-nuclei preparation for scATAC-seq were prepared from day 21 mEB as follows: At day 21 of mEB cultures, mEBs (two 10 cm suspension plates) are collected into a 50 mL conical tube and washed 2x with 1x PBS (without Ca2+, Mg2+). After consecutive PBS washes, mEBs are treated with 1.5mL of 0.25% trypsin and incubated in 37C bath with gentle agitation (steady concentric swirls in 50mL conical tubes) for 3 minutes. For further dissociation, mEBs are then gently triturated 10 times with a P1000 pipette and again incubated at 37C for 3 minutes with gentle agitation. After second incubation, mEBs are gently triturated 10 times with a P1000 pipette. Trypsin digestion is inactivated with CA medium and cells filtered to single-cell suspension through a 100 um filter into a new 50 mL conical. Single cell suspension was counted, and cells spun down at 300 g for 5 minutes. After removing supernatant, wash 1x with 1 mL of 1x PBS + 0.04% BSA and gently pipette mix 5x. Transfer to a 1.5 mL tube and spin at 300g for 5 min at 4C. Wash again with 1mL of 1x PBS + 0.04% BSA. Again, spin at 300 g for 5 min at 4C, and proceeded with 10x Genomics "Nuclei isolation for Single Cell ATAC Sequencing" protocol (V1), with two biological replicates (different mEB differentiation) each with two lanes of 10x (four reactions total).