用Tagmentation Assisted Fragmentation法从K562细胞和果蝇神经母细胞的有限起始材料中提取芯片序列

Junaid  Akhtar; Piyush More; Steffen Albrecht

doi:10.21769/BioProtoc.3520

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Peer-reviewed

ChIP-Seq from Limited Starting Material of K562 Cells and Drosophila Neuroblasts Using Tagmentation Assisted Fragmentation Approach

用Tagmentation Assisted Fragmentation法从K562细胞和果蝇神经母细胞的有限起始材料中提取芯片序列

JA Junaid Akhtar email

PM Piyush More ^*

SA Steffen Albrecht ^*

(*contributed equally to this work) 发布: 2020年02月20日第10卷第4期 DOI: 10.21769/BioProtoc.3520 浏览次数: 3947

评审: Imre GáspárPrashanth N SuravajhalaNarendranath Bhokisham

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Jul 2019

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Abstract

Chromatin immunoprecipitation is extensively used to investigate the epigenetic profile and transcription factor binding sites in the genome. However, when the starting material is limited, the conventional ChIP-Seq approach cannot be implemented. This protocol describes a method that can be used to generate the chromatin profiles from as low as 100 human or 1,000 Drosophila cells. The method employs tagmentation to fragment the chromatin with concomitant addition of sequencing adaptors. The method generates datasets with high signal to noise ratio and can be subjected to standard tools for ChIP-Seq analysis.

Keywords: Low Input ChIP-Seq (低细胞量ChIP-Seq)

Easy workflow (简易工作流程)

High Signal-to-Noise ratio (高信噪比)

Tagmentation (Tagmentation技术)

High reproducibility (重现性好)

Background

Epigenetic state and transcription factors occupancy are critical determinants of gene expression. To understand this regulation, the precise mapping of chromatin features are very important. Chromatin immunoprecipitation coupled with next generation sequencing is a powerful technique that gives valuable insight into genome wide distributions of chromatin features (Ghavi-Helm et al., 2016). Although powerful, this technique is limited in its application owing to the need of significant starting material which in some conditions are hard to meet, if not completely impossible. This limitation has driven many recent efforts to adapt ChIP-Seq approach for low amount starting material or for rare cell types (Adli et al., 2010; Zheng et al., 2015). One of the key limitations of these efforts is the use of sonication or MNAse for fragmenting the chromatin. The former approach is detrimental for the epitope when present in limited amount as it can lead to destruction of the epitope used for subsequent immunoprecipitation (Stathopulos et al., 2004). The MNAse approach on the other hand is hard to control for its efficacy and saturation. The recent application of CUT&RUN approach was successful in generating profiles from samples of 100 cells using antibody-targeted micrococcal nuclease (Skene et al., 2018). However, all these approaches still resort to ligation-based library preparation approach involving multiple steps, potentially leading to increased variability as well as of loss of complexity (Seguin-Orlando et al., 2013). The method described here overcomes these limitations by using commercially available Tn5 enzyme for fragmenting the chromatin with simultaneous preparation of libraries with fewer intermediate steps. The direct comparison of CUT&RUN approach and TAF-ChIP reveals superior signal to noise ratio in the later, making use of standard bioinformatics pipeline amenable to this approach.

Materials and Reagents

DNA Low binding 1.5 ml tubes (Eppendorf, catalog number: 0030108051)
Phase Lock tubes (5Prime, catalog number: 2302830)
Agencourt AMPure XP Beads (Beckmann Coultier, catalog number: A63381)
Protein G Dynabeads (Thermo Fisher Scientific, catalog number: 10003D)
BSA (Sigma-Aldrich, catalog number: A4737)
Proteinase K (Thermo Fisher Scientific, catalog number: E00491)
Yeast tRNA (Sigma-Aldrich, catalog number: R8759)
Anti H3K27Me3 antibody (Active Motif, catalog number: 39155)
Anti H3 antibody (Abcam, catalog number: ab1791)
Anti H3K4Me3 antibody (Abcam, catalog number: ab8580)
Anti H3K9Me3 antibody (Active Motif, catalog number: 39161)
Collagenase I (Sigma-Aldrich, catalog number: 1148089)
Papain (Sigma-Aldrich, catalog number: 1495005)

Primers (Table 1)

Table 1. List of primers with unique barcodes

	Primers
fw ATAC-seq primer, general, no index	AATGATACGGCGACCACCGAGATCTACACTCGTCGGCAGCGTCAGATGT*G
rev ATAC-seq primer, Truseq index, 34 CATGGC	CAAGCAGAAGACGGCATACGAGATGCCATGGTCTCGTGGGCTCGGAGATG*T
rev ATAC-seq primer, Truseq index, 48 TCGGCA	CAAGCAGAAGACGGCATACGAGATTGCCGAGTCTCGTGGGCTCGGAGATG*T
rev ATAC-seq primer, Truseq index, 22 CGTACG	CAAGCAGAAGACGGCATACGAGATCGTACGGTCTCGTGGGCTCGGAGATG*T
rev ATAC-seq primer, Truseq index, 15 ATGTCA	CAAGCAGAAGACGGCATACGAGATTGACATGTCTCGTGGGCTCGGAGATG*T
rev ATAC-seq primer, Truseq index, 46 TCCCGA	CAAGCAGAAGACGGCATACGAGATTCGGGAGTCTCGTGGGCTCGGAGATG*T
rev ATAC-seq primer, Truseq index, 45 TCATTC	CAAGCAGAAGACGGCATACGAGATGAATGAGTCTCGTGGGCTCGGAGATG*T
rev ATAC-seq primer, Truseq index, 40 CTCAGA	CAAGCAGAAGACGGCATACGAGATTCTGAGGTCTCGTGGGCTCGGAGATG*T
rev ATAC-seq primer, Truseq index, 39 CTATAC	CAAGCAGAAGACGGCATACGAGATGTATAGGTCTCGTGGGCTCGGAGATG*T
rev ATAC-seq primer, Truseq index, 38 CTAGCT	CAAGCAGAAGACGGCATACGAGATAGCTAGGTCTCGTGGGCTCGGAGATG*T
rev ATAC-seq primer, Truseq index, 37 CGGAAT	CAAGCAGAAGACGGCATACGAGATATTCCGGTCTCGTGGGCTCGGAGATG*T
rev ATAC-seq primer, Truseq index, 36 CCAACA	CAAGCAGAAGACGGCATACGAGATTGTTGGGTCTCGTGGGCTCGGAGATG*T
rev ATAC-seq primer, Truseq index, 35 CATTTT	CAAGCAGAAGACGGCATACGAGATAAAATGGTCTCGTGGGCTCGGAGATG*T
rev ATAC-seq primer, Truseq index, 25 ACTGAT	CAAGCAGAAGACGGCATACGAGATATCAGTGTCTCGTGGGCTCGGAGATG*T
rev ATAC-seq primer, Truseq index, 26 ATGAG	CAAGCAGAAGACGGCATACGAGATGCTCATGTCTCGTGGGCTCGGAGATG*T
rev ATAC-seq primer, Truseq index, 27 ATTCCT	CAAGCAGAAGACGGCATACGAGATAGGAATGTCTCGTGGGCTCGGAGATG*T
rev ATAC-seq primer, Truseq index, 28 CAAAAG	CAAGCAGAAGACGGCATACGAGATCTTTTGGTCTCGTGGGCTCGGAGATG*T
rev ATAC-seq primer, Truseq index, 23 GAGTGG	CAAGCAGAAGACGGCATACGAGATCCACTCGTCTCGTGGGCTCGGAGATG*T
rev ATAC-seq primer, Truseq index, 24 GGTAGC	CAAGCAGAAGACGGCATACGAGATGCTACCGTCTCGTGGGCTCGGAGATG*T
rev ATAC-seq primer, Truseq index, 42 TAATCG	CAAGCAGAAGACGGCATACGAGATCGATTAGTCTCGTGGGCTCGGAGATG*T
rev ATAC-seq primer, Truseq index, 41 GACGAC	CAAGCAGAAGACGGCATACGAGATGTCGTCGTCTCGTGGGCTCGGAGATG*T

NEXTERA XT DNA Library prep Kit (Illumina, catalog number: FC-131-1024)
NEBNext High-Fidelity 2x PCR Master Mix (New England Biolabs, catalog number: M0541)
Glycogen (Roche, catalog number: 10901393001)
37% Formaldehyde (Sigma-Aldrich, catalog number: F8775)
Glycine (Sigma-Aldrich, catalog number: 50046)
Protease Inhibitor Cocktail (Bimake, catalog number: B14001)
Tris Buffer pH 8.0 (Applichem, catalog number: A4577)
EDTA (Sigma-Aldrich, EDS catalog number: EDS-500G)
Sodium Chloride (Sigma-Aldrich, catalog number: S3014)
Triton X-100 (Sigma-Aldrich, catalog number: T8787)
20% sodium dodecyl sulfate (Sigma-Aldrich, catalog number: 05030)
Nuclease-free water (Qiagen, catalog number: 129115)
Tris (hydroxymethyl) aminomethane (Sigma-Aldrich, catalog number: 252859)
N,N-Dimethylformamide (Sigma-Aldrich, catalog number: D4551)
Qubit dsDNA High Sensitivity Kit (Thermo Fisher Scientific, catalog number: Q33230)
DNA High Sensitivity Kit for Bioanalyzer (Agilent, catalog number: 5067-4627)
Schneider’s medium (Thermo Fisher Scientific, catalog number: 21720-001)
Phenol:Chloroform (Sigma-Aldrich, catalog number: P3803)
3 M sodium acetate (Thermo Fisher Scientific, catalog number: AM9740)
Ethanol (Sigma-Aldrich, catalog number: 51976)
Hoechst stain (Thermo Fisher Scientific, catalog number: 62249)
MgCl₂ (Sigma-Aldrich, catalog number: 63068)
Dissociation Solution (see Recipes)
Blocking Buffer (see Recipes)
RIPA (140 mM) (see Recipes)
RIPA (250 mM) (see Recipes)
TE Buffer (see Recipes)
Tagmentation Buffer (Home-made) (see Recipes)

Equipment

DiaMag 0.2 ml-magnetic rack (Diagenode, catalog number: B04000001)
DynaMag-2 magnetic rack (Thermo Fisher Scientific, catalog number: 12321D)
Bioruptor plus (Diagenode, catalog number: B01020001)
Thermomix compact (Eppendorf, catalog number: T1317)
Bio-Rad PCR Cycler (Bio-Rad, catalog number: C1000)
Mini tube rotator (Biotools, AG, R2001)
Tabletop minicentrifuge (Thermo Fisher Scientific, catalog number: 75002478)
Agilent Bioanalyzer 2100 (Agilent, catalog number: G2939BA)
Qubit Fluorometer 3 (Thermo Fisher Scientific, catalog number: Q33216)
Flow cytometer (BD FACS Aria)

Software

MACS2 (version 2.1.1.20160309, https://github.com/taoliu/MACS)
Deeptools (version 3.1.3, https://github.com/deeptools/deepTools)
Samtools (version 1.09, https://github.com/samtools/samtools)
bowtie2 (version 2.3.5, https://github.com/BenLangmead/bowtie2)
FastQC (version 0.10.1, https://github.com/s-andrews/FastQC)

Procedure

English

中文翻译

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如何引用

Akhtar, J., More, P. and Albrecht, S. (2020). ChIP-Seq from Limited Starting Material of K562 Cells and Drosophila Neuroblasts Using Tagmentation Assisted Fragmentation Approach. Bio-protocol 10(4): e3520. DOI: 10.21769/BioProtoc.3520.