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An Integrated Workflow to Study the Promoter-Centric Spatio-Temporal Genome Architecture in Scarce Cell Populations

Published: April 21st, 2023



1Josep Carreras Leukaemia Research Institute, 2Barcelona Supercomputing Center, 3Catalan Institution for Research and Advanced Studies (ICREA), 4Institute for Health Science Research Germans Trias i Pujol
* These authors contributed equally

Gene expression is regulated by interactions of gene promoters with distal regulatory elements. Here, we descirbe how low input Capture Hi-C (liCHi-C) allows the identification of these interactions in rare cell types, which were previously unmeasurable.

Spatiotemporal gene transcription is tightly regulated by distal regulatory elements, such as enhancers and silencers, which rely on physical proximity with their target gene promoters to control transcription. Although these regulatory elements are easy to identify, their target genes are difficult to predict, since most of them are cell-type specific and may be separated by hundreds of kilobases in the linear genome sequence, skipping over other non-target genes. For several years, Promoter Capture Hi-C (PCHi-C) has been the gold standard for the association of distal regulatory elements to their target genes. However, PCHi-C relies on the availability of millions of cells, prohibiting the study of rare cell populations such as those commonly obtained from primary tissues. To overcome this limitation, low input Capture Hi-C (liCHi-C), a cost-effective and customizable method to identify the repertoire of distal regulatory elements controlling each gene of the genome, has been developed. liCHi-C relies on a similar experimental and computational framework as PCHi-C, but by employing minimal tube changes, modifying the reagent concentration and volumes, and swapping or eliminating steps, it accounts for minimal material loss during library construction. Collectively, liCHi-C enables the study of gene regulation and spatiotemporal genome organization in the context of developmental biology and cellular function.

Temporal gene expression drives cell differentiation and, ultimately, organism development, and its alteration is closely related to a wide plethora of diseases1,2,3,4,5. Gene transcription is finely regulated by the action of regulatory elements, which can be classified as proximal (i.e., gene promoters) and distal (e.g., enhancers or silencers), the latter of which are frequently located afar from their target genes and physically interact with them through chromatin looping to modulate gene expression

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To ensure minimal material loss, (1) work with DNA low-binding tubes and tips (see Table of Materials), (2) place reagents on the tube wall instead of introducing the tip inside the sample and, (3) if possible, mix the sample by inversion instead of pipetting the sample up and down, and spin down afterward to recover the sample.

1. Cell fixation

  1. Cells growing in suspension
    1. Harvest 50,000 to one million cells and place them in a DNA low-.......

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liCHi-C offers the possibility of generating high-quality and resolution genome-wide promoter interactome libraries with as little as 50,000 cells53. This is accomplished by – besides the drastic reduction of reaction volumes and the use of DNA low-binding plasticware throughout the protocol – removing unnecessary steps from the original protocol, in which significant material losses occur. These include the phenol purification after decrosslinking, the biotin removal, and subsequ.......

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liCHi-C offers the capability of generating high-resolution promoter interactome libraries using a similar experimental framework from PCHi-C's but with a vastly reduced cell number. This is greatly achieved by eliminating unnecessary steps, such as phenol purification and biotin removal. In the classical in-nucleus ligation Hi-C protocol57 and its subsequent derivative technique PCHi-C, biotin is removed from non-ligated restriction fragments to avoid pulling down DNA fragments that are after.......

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We thank the rest of the members from the Javierre lab for their feedback on the manuscript. We thank CERCA Program, Generalitat de Catalunya, and the Josep Carreras Foundation for institutional support. This work was financed by FEDER/Spanish Ministry of Science and Innovation (RTI2018-094788-A-I00), the European Hematology Association (4823998), and the Spanish Association against Cancer (AECC) LABAE21981JAVI. BMJ is funded by La Caixa Banking Foundation Junior Leader project (LCF/BQ/PI19/11690001), LR is funded by an AGAUR FI fellowship (2019FI-B00017), and LT-D is funded by an FPI Fellowship (PRE2019-088005). We thank the biochemistry and molecular biology PhD pro....

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NameCompanyCatalog NumberComments
0.4 mM Biotin-14-dATPInvitrogen19524-016
0.5 M EDTA pH 8.0InvitrogenAM9260G
1 M Tris pH 8.0InvitrogenAM9855G
10x NEBuffer 2New England BiolabsB7002SReferenced as restriction buffer 2 in the manuscript
10x PBSFisher ScientificBP3994
10x T4 DNA ligase reaction bufferNew England BiolabsB0202S
16% formaldehyde solution (w/v), methanol-freeThermo Scientific28908
20 mg/mL Bovine Serum AlbuminNew England BiolabsB9000S
5 M NaClInvitrogenAM9760G
5PRIME Phase Lock Gel Light tubesQiuantabio2302820For phenol-chloroform purification in section 4 (DNA purification). Phase Lock Gel tubes are a commercial type of tubes specially designed to maximize DNA recovery after phenol-chloroform purifications while avoiding carryover of contaminants in the organic phase by containing a resin of intermediate density which settles between the organic and aqueous phase and isolates them. PLG tubes should be spun at 12,000 x g for 30 s before use to ensure that the resin is well-placed at the bottom of the tube
Adapters and PCR primers for library amplificationIntegrated DNA Technologies-Bought as individual primers with PAGE purification for NGS
Cell scrappersNunc179693Or any other brand
Centrifuge (fixed-angle rotor for 1.5 mL tubes)Any brand
CHiCAGO R package1.14.0
CleanNGS beadsCleanNACNGS-0050
dATP, dCTP, dGTP, dTTPPromegaU120A, U121A, U122A, U123AOr any other brand
DNA LoBind tube, 1.5 mLEppendorf30108051
DNA LoBind tube, 2 mLEppendorf30108078
DNA polymerase I large (Klenow) fragment 5000 units/mLNew England BiolabsM0210L
Dynabeads MyOne Streptavidin C1 beadsInvitrogen65002For biotin pull-down of the pre-captured library in section 8 (biotin pull-down)
Dynabeads MyOne Streptavidin T1 beadsInvitrogen65602For biotin pull-down of the post-captured library in section 11 (biotin pull-down and PCR amplification)
DynaMag-2Invitrogen12321DOr any other magnet suitable for 1.5 ml tubeL
Ethanol absoluteVWR20821.321
FBS, qualifiedGibco10270-106Or any other brand
GlycineFisher BioReagentsBP381-1
GlycoBlue CoprecipitantInvitrogenAM9515Used for DNA coprecipitation in section 4 (DNA purification)
HindIII, 100 U/µLNew England BiolabsR0104T
IGEPAL CA-630Sigma-AldrichI8896-50ML
Klenow EXO- 5000 units/mLNew England BiolabsM0212L
Low-retention filter tips (10 µL, 20 µL, 200 µL and 1000 µL)ZeroTipPMT233010, PMT252020, PMT231200, PMT252000
M220 Focused-ultrasonicatorCovaris500295
Micro TUBE AFA Fiber Pre-slit snap cap 6 x 16 mm vialsCovaris520045For sonication in section 6 (sonication)
NheI-HF, 100 U/µLNew England BiolabsR3131M
Nuclease-free molecular biology grade waterSigma-AldrichW4502
PCR primers for quality controlsIntegrated DNA Technologies-
PCR strips and capsAgilent Technologies410022, 401425
Phenol: Chloroform: Isoamyl Alcohol 25:24:1, Saturated with 10 mM Tris, pH 8.0, 1 mM EDTASigma-AldrichP3803
Phusion High-Fidelity PCR Master Mix with HF BufferNew England BiolabsM0531LFor amplification of the library in sections 9 (dATP-tailing, adapter ligation and PCR amplification)
and 11 (biotin pull-down and PCR amplification)
Protease inhibitor cocktail (EDTA-free)Roche11873580001
Proteinase K, recombinant, PCR gradeRoche3115836001
Qubit 1x dsDNA High Sensitivity kitInvitrogenQ33230For DNA quantification after precipitation in section 4 (DNA purification)
Qubit assay tubesInvitrogenQ32856
rCutsmart bufferNew England BiolabsB6004S
RPMI Medium 1640 1x + GlutaMAXGibco61870-010Or any other brand
SDS - Solution 10% for molecular biologyPanReac AppliChemA0676
Sodium acetate pH 5.2Sigma-AldrichS7899-100ML
SureSelect custom 3-5.9 Mb libraryAgilent Technologies5190-4831Custom designed mouse or human capture system, used for the capture
SureSelect Target Enrichment Box 1Agilent Technologies5190-8645Used for the capture
SureSelect Target Enrichment Kit ILM PE Full AdapterAgilent Technologies931107Used for the capture
T4 DNA ligase 1 U/µLInvitrogen15224025For ligation in section 3 (ligation and decrosslink)
T4 DNA ligase 2000000/mLNew England BiolabsM0202TFor ligation in section 9 (dATP-tailing, adapter ligation and PCR amplification)
T4 DNA polymerase 3000 units/mLNew England BiolabsM0203L
T4 PNK 10000 units/mLNew England BiolabsM0201L
Tapestation 4200 instrumentAgilent TechnologiesFor automated electrophoresis in section 9 (dATP-tailing, adapter ligation, and PCR amplification) and
section 11
(Biotin pull-down and PCR amplification). Any other automated electrophoresis system is valid
Tapestation reagentsAgilent Technologies5067-5582, 5067-5583, 5067-5584, 5067-5585,For automated electrophoresis in section 9 (dATP-tailing, adapter ligation, and PCR amplification) and
section 11
(Biotin pull-down and PCR amplification). Any other automated electrophoresis system is valid
Triton X-100 for molecular biologyPanReac AppliChemA4975
Tween 20Sigma-AldrichP9416-50ML

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