使用 MapR对果蝇中的 R 环进行组织特异性、全基因组映射

Juan Jauregui-Lozano; Kendall Cottingham; Hana Hall

doi:10.21769/BioProtoc.4516

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Peer-reviewed

Tissue-Specific, Genome-wide Mapping of R-loops in Drosophila Using MapR

使用 MapR对果蝇中的 R 环进行组织特异性、全基因组映射

JJ Juan Jauregui-Lozano email

KC Kendall Cottingham

HH Hana Hall

发布: 2022年09月20日第12卷第18期 DOI: 10.21769/BioProtoc.4516 浏览次数: 1517

评审: Giusy TornilloMario RuizNingfei An

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Cover of Aging Cell, featuring study using the protocol.

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Abstract

R-loops, or RNA:DNA hybrids, are structures that arise co-transcriptionally when a nascent RNA hybridizes back with the template ssDNA, leading to a displaced ssDNA. Because accumulation of R-loops can lead to genomic instability and loss of cellular homeostasis, it is important to determine the genome-wide distribution of R-loops in different physiological conditions. Current R-loop mapping strategies are based on R-loop enrichment—mediated by the S9.6 antibody, such as DRIP-seq, or by the exonuclease RNase H1, such as MapR—or the latest R-loop CUT&Tag, based on an artificial R-loop sensor derived from an RNase H1 sub-domain. Because some of these techniques often require high input material or expensive reagents, we sought to apply MapR, which does not require expensive reagents and has been shown to be compatible with low input samples. Importantly, we demonstrate that incorporation of improved CUT&RUN steps into the MapR protocol yields R-loop-enriched DNA when using low input Drosophila nuclei.

Graphical abstract:

Workflow for mapping tissue-specific, genome-wide R-loops in Drosophila. Purify GST-tagged and catalytically inactive RNase H1 tethered MapR enzymes, GST-ΔRH-MNase, and GST-MNase, from transformed E. coli. Perform tissue-specific nuclei immuno-enrichment from UAS-EGFP.KASH-Msp300 Drosophila using magnetic bead–bound green fluorescent protein (GFP) antibody. Incubate isolated nuclei with MapR enzymes and activate MNase DNA cleavage with low salt/high calcium buffers. Purify released, R-loopenriched DNA fragments and generate sequencing-ready libraries. Align MapR data to reference genome and compare R-loop enrichment peaks in genome browser.

Keywords: R-loop (R环)

Drosophila (果蝇)

Bioinformatics (生物信息学)

MapR (MapR)

Neurons (神经元)

Background

R-loops are RNA:DNA hybrids that mostly form during transcription as a result of a nascent RNA strand annealing to a template DNA strand while misplacing a single-stranded DNA. R-loops play an important role in biological processes such as class switch recombination and mitochondrial replication. However, persistent or un-scheduled R-loop formation is a major source of spontaneous DNA damage that can lead to genome instability, one of the key hallmarks of aging (García-Muse and Aguilera, 2019). R-loop resolution is particularly important for neuronal function, as mutations in the R-loop processing factors are associated with neurodegenerative disorders such as Aicardi–Goutières syndrome, ataxia with oculomotor apraxia, and amyotrophic lateral sclerosis (Becherel et al., 2015; Lim et al., 2015; Salvi and Mekhail, 2015). Strategies to map R-loops are based on using either the S9.6 antibody, the RNase H1 enzyme, or more recently, the RNase H1–derived N-terminal hybrid-binding domain (HBD) fused to a Tn5 (Wang et al., 2021). However, the majority of these methods can require high amounts of starting material, or extensive and expensive protocols.

Here, we modified a recently published method, called MapR, that uses the catalytically inactive ribonuclease H1 (RNase H1) enzyme to enrich for R-loops. MapR, which is based on CUT&RUN, utilizes an RNase H1 tethered to a micrococcal nuclease (MNase) for cleavage and release of R-loop-enriched DNA (Yan and Sarma, 2020). Because using the traditional MapR protocol with low input Drosophila samples led to over-digested DNA (Jauregui-Lozano et al., 2022), we adapted the MapR protocol with steps from an improved published CUT&RUN protocol, which incorporates low salt, high calcium buffers to decrease MNase activation time, as well as background binding (Meers et al., 2019). The main advantage of this approach is the ability to map R-loops where starting material is very limited, such as specific tissues or cells from the whole animal. Using this method, we show that Drosophila melanogaster photoreceptor (PR) neurons accumulate R-loops during aging, which correlates with decreased expression of genes with neuronal function and decreased visual function (Jauregui-Lozano et al., 2022). Thus, studies that combine cell-specific approaches provide us with a unique and powerful tool to map R-loops genome-wide and study R-loop-associated toxicity in the context of neuronal aging.

Materials and Reagents

5 or 15 mL conical tube
40 µm cell strainer (Corning, catalog number: 431750)
Slide-A-Lyzer^TM G2 dialysis cassettes, 10K MWCO, 3 mL (Thermo Fisher, catalog number: 87730)
Pierce^TM glutathione magnetic agarose beads (Thermo Fisher, catalog number: 78601). Keep at 4 °C
One Shot BL21 (DE3) chemically competent E. coli (Thermo Fisher, catalog number: C600003). Keep unused E. coli pellets at -80 °C
L-Glutathione reduced (Millipore Sigma, catalog number: G4251-300MG)
HyperPAGE II pre-stained protein marker (Bioline, catalog number: BIO-33066). Keep aliquots at -20 °C
Dynabeads^TM protein G for immunoprecipitation (Invitrogen, catalog number: 10003D). Keep at 4 °C
Anti-GFP antibody (Sigma-Aldrich, catalog number: 11814460001). Keep at -20 °C upon resuspension
UAS-EGFP.KASH-Msp300 flies (Bloomington Drosophila Stock Center, BDSC#92580)
Digitonin 5% (Thermo Fisher, catalog number: BN2006)
UltraPure^TM dithiothreitol (DTT) 0.1 M solution (Thermo Scientific, catalog number 707265ML)
UltraPure^TM ethylenediaminetetraacetic acid, disodium salt, dihydrate (EDTA) (Thermo Fisher, catalog number: 15576028)
UltraPure^TM 1 M Tris-HCl, pH 8.0 (Invitrogen, catalog number 15568025)
EGTA, molecular biology grade (Millipore, catalog number: 324626)
cOmplete^TM, mini, EDTA-free protease inhibitor cocktail tablets (Roche, catalog number: 04693159001)
Corning^® 100 mL HEPES, liquid 1 M solution (238.3 mg/mL) (Corning, catalog number: 25-060-CI)
IGEPAL^® CA-630 (Supelco, catalog number: 56741-50ML-F)
TWEEN^® 20 (Sigma-Aldrich, catalog number: P1379)
Coomassie Brilliant Blue R-250 dye (Thermo Scientific, catalog number: 20278)
Acetic acid glacial, ReagentPlus^® (Sigma-Aldrich, catalog number A6283)
Methanol, ACS reagent (Sigma-Aldrich, catalog number 179337)
Spermidine (Sigma-Aldrich, catalog number: S022-1G)
RNase A (Thermo Fisher, catalog number: 12091021)
Linear acrylamide (Thermo Fisher, catalog number: AM9520)
RNase H (New England BioLabs, catalog number M0297S). Keep at -20 °C
ChIP DNA Clean & Concentrator (Zymo, catalog number D5205)
Qubit^TM 1× dsDNA high sensitivity (HS) (Thermo Fisher, catalog number: Q33230)
Note: We recommend using high sensitivity assays since obtained DNA can be low-input and difficult to quantify using traditional reagents.
Ovation^® ultralow V2 DNA-Seq library preparation kit (Tecan, catalog number: 0344NB-08)
GST-wash/equilibration buffer (see Recipes)
GST-elution buffer (see Recipes)
Coomassie fixing solution (see Recipes)
Coomassie staining solution (see Recipes)
Coomassie destaining solution (see Recipes)
Homogenization/wash buffer (see Recipes)
Dilution buffer (see Recipes)
Bead washing buffer (see Recipes)
PBST (1×, pH 7.4) (see Recipes)
Dig-wash buffer (see Recipes)
Low salt rinse buffer (see Recipes)
Activation buffer (see Recipes)
EGTA-STOP buffer (1×) (see Recipes)

Equipment

Branson sound amplifier
Agilent TapeStation 4200
WHEATON^® Dounce tissue grinder, 1 mL
37 °C incubator with shaking platform (such as Thomas Scientific SCO2W benchtop water jacketed CO₂ incubator, catalog number: 1229P58)
Cold (4 °C) centrifuge (such as Eppendorf^TM, model: Centrifuge 5424 R)
Cold (4 °C) incubator (such as Fisherbrand^TM mini low temperature refrigerated incubator, catalog number: 15-015-2632)
Tube revolver rotator (such as Thermo Scientific^TM tube revolver rotator, catalog number: 88881001)
Magnetic rack (such as Millipore PureProteome^TM magnetic stand, catalog number: LSKMAGS08)
Gel electrophoresis chamber (such as Bio-Rad Mini-PROTEAN^® tetra vertical electrophoresis cell, catalog number: 1658025FC)

Software

Genomic alignment of sequencing reads: Bowtie2 (Langmead and Salzberg, 2012)
BAM file processing: Samtools (Li et al., 2009)
Bigwig generation/processing: Deeptools (Ramírez et al., 2014)
Integrative genome browser (Thorvaldsdóttir et al., 2013)

Procedure

English

中文翻译

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如何引用

Jauregui-Lozano, J., Cottingham, K. and Hall, H. (2022). Tissue-Specific, Genome-wide Mapping of R-loops in Drosophila Using MapR. Bio-protocol 12(18): e4516. DOI: 10.21769/BioProtoc.4516.