RNASeq in C. elegans Following Manganese Exposure

Nancy L. Parmalee1, Shahina B. Maqbool2, Bin Ye3, Brent Calder3, Aaron B. Bowman4, Michael Aschner1

1 Department of Molecular Pharmacology, Albert Einstein College of Medicine, New York City, New York, 2 Epigenomics Shared Facility, Albert Einstein College of Medicine, New York City, New York, 3 Computational Genomics Core, Albert Einstein College of Medicine, New York City, New York, 4 Department of Neurology, Vanderbilt University, Nashville, Tennessee
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 11.20
DOI:  10.1002/0471140856.tx1120s65
Online Posting Date:  August, 2015
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Manganese is a metal that is required for optimal biological functioning of organisms. Absorption, cellular import and export, and excretion of manganese are all tightly regulated. While some genes involved in regulation, such as DMT‐1 and ferroportin, are known, it is presumed that many more are involved and as yet unknown. Excessive exposure to manganese, usually in industrial settings such as mining or welding, can lead to neurotoxicity and a condition known as manganism that closely resembles Parkinson's disease. Elucidating transcriptional changes following manganese exposure could lead to the development of biomarkers for exposure. This unit presents a protocol for RNA sequencing in the worm Caenorhabditis elegans to assay for transcriptional changes following exposure to manganese. This protocol is adaptable to any environmental exposure in C. elegans. The protocol results in counts of gene transcripts in control versus exposed conditions and a ranked list of differentially expressed genes for further study. © 2015 by John Wiley & Sons, Inc.

Keywords: manganese; C. elegans; RNASeq

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Table of Contents

  • Introduction
  • Basic Protocol 1: Synchronization of Worms and Mn Treatment
  • Basic Protocol 2: RNA Isolation and Quantification
  • Basic Protocol 3: Library Preparation and Sequencing
  • Basic Protocol 4: Alignment and Analysis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
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Basic Protocol 1: Synchronization of Worms and Mn Treatment

  • N2 strain C. elegans or other strain of interest
  • 8 P agar (see recipe) in 150‐mm plates (Corning, cat no. 351058)
  • M9 buffer (see recipe) Hypochlorite solution (see recipe)
  • 30% (w/v) sucrose (Fisher, cat. no. S5‐3) in H 2O
  • NGM agar (see recipe) in 35‐mm plates (Corning, cat. no. 351008)
  • E. coli strain NA22
  • Manganese chloride tetrahydrate (MnCl 2·4H 2O; Sigma Aldrich cat. no. 203734‐25 G)
  • 85 mM NaCl in commercial‐grade H 2O (Fisher, cat no. BP358‐212)
  • Commercial‐grade H 2O (Fisher, cat. no. BP24854
  • E. coli strain OP50
  • Dissecting microscope (Zeiss Stemi 2000)
  • 37°C bacterial incubator
  • 15‐ml conical tubes (Crystalgen cat. no. 23‐2266) Centrifuge
  • Nutator (Spectrum Biomixer)
  • 150‐mm petri plates (Corning, cat no. 351058)
  • Microscope slides
  • Siliconized microcentrifuge tubes (Denville, cat. no. C19035)
  • 35‐mm petri plates (Corning, cat. no. 351008)
  • Additional reagents and equipment for growing C. elegans (Brenner, )

Basic Protocol 2: RNA Isolation and Quantification

  • C. elegans treated as in protocol 1, steps 1 to 25
  • Trizol reagent (Life Technologies, cat. no. 15596018)
  • Liquid N 2
  • Chloroform (EMD, cat. no. CX‐1059‐1)
  • RNaseZap (Life Technologies, cat. no. AM9780)
  • Nuclease‐free water (Life Technologies, cat. no. AM9932)
  • Isopropanol (EMD, cat. no. PX‐1834P‐1)
  • Glycogen (Life Technologies cat. no. AM9510)
  • 75% ethanol prepared with RNase‐free H 2O
  • Turbo DNA‐free DNase kit (Life Technologies, cat. no. AM1907)
  • MinElute Cleanup kit (Qiagen, cat. no. 74204)
  • SureOne 100‐ to 1025‐μl pipet tips (Fisher, cat. no. 02‐707‐408), or equivalent
  • NanoDrop 2000 spectrophotometer (NanoDrop, Thermo Scientific)
  • Agilent 2100 Bioanalyzer (Agilent Technologies)

Basic Protocol 3: Library Preparation and Sequencing

  • High‐quality total RNA from C. elegans ( protocol 2)
  • Ribo‐Zero Magnetic Gold Kit (Human/Mouse/Rat; Epicentre‐Illumina, cat. no. MRZG12324)
  • Ethachinmate (ECM); Wako, cat. no. 312‐01791)
  • Nuclease‐free water (Life Technologies, cat. no. AM9932)
  • Qubit RNA BR Assay Kit (Invitrogen, cat. no. Q10210)
  • ERCC Ex fold Spike‐In mix2 (Life Technologies, cat. no. 4456739; optional)
  • SuperScript III First‐Strand Synthesis System (Invitrogen, cat. no. 18080‐051)
  • Actinomicyn D (Sigma, cat. no. A1410‐2MG or Fisher, cat. no. BP60610); working concentration, 125 ng/μl in H 2O
  • RNaseOUT (Invitrogen, cat no. 10777‐019)
  • 3 M sodium acetate, pH 5.5
  • 100% and 70% ethanol
  • 5× second‐strand buffer (Invitrogen, cat. no. 10812‐014)
  • dUTP set (100 mM each of dATP, dCTP, dGTP, dUTP; Promega, cat. no. U‐1335)
  • E. coli DNA ligase (Invitrogen, cat. no. 18052‐019)
  • E. coli DNA polymerase (Invitrogen, cat. no. 18010‐025)
  • MinElute PCR Purification Kit (Qiagen, cat. no. 28004)
  • QIAQuick gel extraction kit (Qiagen, cat. no. 28704)
  • Low TE buffer: 10 mM Tris·Cl, pH 8.0 to 8.5 ( appendix 2A) with 0.1 mM EDTA
  • KAPA HTP or LTP Library Preparation Kit for Illumina (KAPA Biosystems)
  • TruSeq DNA HT or LTP Sample Prepration Kit for Illumina (Illumina, cat. no. FC‐121‐2003)
  • Agencourt AMPure XP reagent (Beckman Coulter Inc., cat. no. A63882)
  • 1 U/μl uracil‐N‐glycosylase (UNG; Fermentas, cat. no. EN0361)
  • 100 mM dNTPs (Invitrogen, cat. no. 10297‐018)
  • PCR‐grade H 2O
  • PCR Oligo 1 and 2 (Illumina Trueseq; IDT synthesized)
  • 1 U/μl KAPA HiFi Hotstart DNA Polymerase; KAPA Biosystems, cat. no. KK2501)
  • Library Quantification Kit (KAPA, cat. no. KK4854)
  • High Sensitivity DNA Kit for bioanalyzer (Agilent, cat. no. 5067‐4626)
  • Agilent 2100 Bioanalyzer (Agilent Technologies)
  • RNA Pico chip (Agilent)
  • Heat block
  • Thermal cycler (BioRad)
  • Covaris S2 High Performance Ultrasonicator (Covaris, Inc.)
  • Qubit dsDNA HS Assay Kit (Invitrogen, cat. no. Q32851)
  • LightCycler 480 Multiwell Plate, 384‐well, white (Roche, cat. no. 04729749001)
  • Light Cycler 480 qPCR instrument (Roche)
  • Additional reagents and equipment for agarose gel electrophoresis (Voytas, )

Basic Protocol 4: Alignment and Analysis

  • gsnap, v2012‐07‐20 (Linux)
  • samtools, v0.1.19 (Linux)
  • HTseq, v0.5.3p3 (Linux)
  • R, v3.1.0 (platform independent)
  • edgeR, v3.6.8 (platform independent)
NOTE: Steps 1 to 3 should be run for each sample, then htseq‐count output for all samples are combined into one file before statistical analysis.
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