蛔虫线虫的细胞核分离

Yuanyuan Kang; Jianbin Wang; Richard E. Davis

doi:10.21769/BioProtoc.2262

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Peer-reviewed

Nuclei Isolation from Nematode Ascaris

蛔虫线虫的细胞核分离

YK Yuanyuan Kang

JW Jianbin Wang

RD Richard E. Davis email

发布: 2017年05月05日第7卷第9期 DOI: 10.21769/BioProtoc.2262 浏览次数: 9020

评审: Gal HaimovichManish ChamoliAnonymous reviewer(s)

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The authors used this protocol in:

Cover of Cell Reports, featuring study using the protocol.

Aug 2016

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Abstract

Preparing nuclei is necessary in a variety of experimental paradigms to study nuclear processes. In this protocol, we describe a method for rapid preparation of large number of relatively pure nuclei from Ascaris embryos or tissues that are ready to be used for further experiments such as chromatin isolation and ChIP-seq, nuclear RNA analyses, or preparation of nuclear extracts (Kang et al., 2016; Wang et al., 2016).

Keywords: Nuclei isolation (核分离)

Background

Nuclei isolation is often the first step in studying the molecular and biochemical aspects of nuclear events. Several methods have been developed to isolate nuclei from different tissues and cell types. However, few nuclei isolation protocols from nematodes other than C. elegans have been described (Ooi et al., 2010; Zanin et al., 2011; Haenni et al., 2012). Embryos of the parasitic nematode Ascaris have been used to prepare a variety of extracts for in vitro cell-free systems (Cohen et al., 2004; Lall et al., 2004). However, these extracts were typically whole cell extracts. Here we describe a method for preparation of nuclei from the nematode Ascaris.

Materials and Reagents

15 ml Falcon tubes (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 339651 )
50 ml Falcon tubes (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 339653 )
225 ml Falcon bottles (Corning, Falcon^®, catalog number: 352075 )
Ascaris
Ascaris can be collected from slaughterhouses that process several thousand pigs a day and use the small intestines to make sausage casings. Ascaris can be picked up by hands (with gloves) from contents of intestine, which are pushed out to a tank by machines. Usually, it takes two persons 3-5 h to collect ~1,000 worms. Both fresh tissues and embryos can be obtained from these live worms. Female Ascaris can also be ordered from Carolina Biological, Living Zoology Department. These females are shipped dead on ice and the Ascaris zygotes can be obtained from the proximal uteri
Sodium hydroxide (NaOH) (Fisher Scientific, catalog number: S318-1 )
H₂O (purified by Milli-Q^® Integral Water Purification System)
DAPI (Santa Cruz Biotechnology, catalog number: sc-3598 )
Liquid nitrogen (Airgas, catalog number: NI NF160LT22 )
Phenylmethanesulfonyl fluoride (PMSF) (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 36978 )
cOmplete^TM protease inhibitor cocktail (Roche Diagnostics, catalog number: 0493116001 )
Sodium chloride (NaCl) (Fisher Scientific, catalog number: S271-3 )
Potassium chloride (KCl) (Fisher Scientific, catalog number: P217-500 )
Sodium phosphate dibasic (Na₂HPO₄) (Fisher Scientific, catalog number: BP350-500 )
Potassium phosphate monobasic (KH₂PO₄) (Fisher Scientific, catalog number: P285-500 )
Hydrochloric acid (HCl)
1 M Tris-HCl, pH 8.0 (Thermo Fisher Scientific, Ambion^TM, catalog number: AM9856 )
1 M Tris-HCl, pH 7.0 (Thermo Fisher Scientific, Ambion^TM, catalog number: AM9851 )
Sodium hypochlorite solution (Fisher Scientific, catalog number: SS290-1 )
Potassium hydroxide (KOH) (Fisher Scientific, catalog number: P250-1 )
Magnesium chloride (MgCl₂) (Fisher Scientific, catalog number: M33-500 )
EGTA (Thermo Fisher Scientific, USB, catalog number: 15703 )
Sucrose (Fisher Scientific, catalog number: BP220-212 )
DTT (Fisher Scientific, catalog number: BP172-5 )
Spermine (Sigma-Aldrich, catalog number: S3256 )
Spermidine (Sigma-Aldrich, catalog number: S2626 )
Nonidet P-40 (NP-40) (Alfa Aesar, Affymetrix/USB, catalog number: J19628 )
Triton X-100 (Promega, catalog number: H5142 )
Glycerol (Fisher Scientific, catalog number: BP229-1 )
2-mercaptoethanol (Sigma-Aldrich, catalog number: M6250-10ML )
EDTA (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 17892 )
PBS buffer (see Recipes)
1 M Tris-HCl, pH 7.8 (see Recipes)
KOH/Bleach (0.4 N KOH/1.4% sodium hypochlorite) (see Recipes)
Nuclei extraction buffer A (see Recipes)
Nuclei extraction buffer B (see Recipes)
Nuclei extraction buffer C (see Recipes)
Nuclei storage buffer (see Recipes)

Equipment

Erlenmeyer flask (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 4112-0125 , 4112-0250 or 4112-0500 )
1 L beaker (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 12011000 )
4 L plastic beaker (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 12014000 )
Shaking incubator (example, Lab-Line, model: 4629 )
Fluorescence microscope (example, Nikon Instruments, model: TS100 )
Refrigerated centrifuge (example, Thermo Fisher Scientific, model: Sorvall^TM Legend^TM XTR )
Metal dounce homogenizer (WHEATON, catalog number: 357574 )
Mortar and pestle (example, Fisher Scientific, catalog numbers: S02591 and S02594 )