Genome-wide Surveillance of Transcription Errors in Eukaryotic Organisms

Summary

This protocol provides researchers with a new tool to monitor the fidelity of transcription in multiple model organisms.

Abstract

Accurate transcription is required for the faithful expression of genetic information. Surprisingly though, little is known about the mechanisms that control the fidelity of transcription. To fill this gap in scientific knowledge, we recently optimized the circle-sequencing assay to detect transcription errors throughout the transcriptome of Saccharomyces cerevisiae, Drosophila melanogaster, and Caenorhabditis elegans. This protocol will provide researchers with a powerful new tool to map the landscape of transcription errors in eukaryotic cells so that the mechanisms that control the fidelity of transcription can be elucidated in unprecedented detail.

Introduction

The genome provides a precise biological blueprint of life. To implement this blueprint correctly, it is important for the genome to be transcribed with great precision. However, transcription is unlikely to be error free. For example, RNA polymerases have long been known to be error-prone in vitro^1,2, and recently it was shown that they commit errors in vivo as well^3,5,6, particularly when confronted with DNA damage^7,8,

Protocol

1. Preparation

1. RNases are omnipresent; therefore, clean the workspace thoroughly by spraying it down with a decontamination reagent (e.g., RNase AWAY) and 70% ethanol. Spray down any pipettes, pens, or tube racks to remove potential sources of RNase contamination.
2. To further prevent RNases from contaminating the samples, wear a lab coat or long-sleeve T-shirt during the experimentation. Avoid contact between the gloves and the inside of any tubes that will be used, especially when retrieving them from a box or bag.
3. Prior to the reverse transcription, change gloves frequently.
   NOTE: For optimal results, d....

Results

Like all massively parallel sequencing approaches, each C-seq experiment produces an unwieldy, large dataset. For first-time users, it can be difficult to handle these datasets; thus, it is recommended that all users contact an experienced bio-informatician prior to the experimentation. On average, the expectation is that users will generate approximately 55–70 Giga bases (Gbases) per run on most massively parallel sequencing platforms. For this protocol, typically, 12–30 samples were mul.......

Discussion

Here, we describe an optimized protocol for the preparation of C-seq libraries for the detection of transcription errors in Saccharomyces cerevisiae, Drosophila melanogaster, and Caenorhabditis elegans. This protocol has numerous advantages over existing protocols, as well as alternative techniques.

Over the past 15 years, numerous reporter systems have been developed that rely on luciferase^7,8 or Cre-Lox rec.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
RiboPure RNA purification kit	ThermoFisher	AM1926	Total RNA purification
Genelute mRNA purification kit	Sigma-Aldrich	MRN70-1KT	mRNA purification
Nuclease-free Water	Ambion	AM9937	Elution and dilution
Ambion RNase III	ThermoFisher	AM2290	RNA fragmentation
T4 RNA Ligase 1 (ssRNA Ligase)	New England Biolabs	M0204S	RNA circularization
Ribolock	ThermoFisher	EO0381	RNase inhibitor
SuperScript III Reverse Transcriptase	ThermoFisher	18080044	Rolling circle reverse transcription
10 mM dNTP mix	ThermoFisher	18427013	Rolling circle reverse transcription
Random hexamers (50 ng/µL)	ThermoFisher	N8080127	Rolling circle reverse transcription
NEB Second Strand Synthesis Module	New England Biolabs	E6111S	Second Strand Synthesis
NEBNext Ultra DNA Library Prep Kit for Illumina	New England Biolabs	E7370S	cDNA library preparation
NEB Next index primers	NEB	E7335S	Multiplex PCR primers
Oligo Clean & Concentrator	Zymo Research	D4061	Clean up of RNA and DNA samples
DynaMag-2 Magnet	ThermoFisher	12321D	Magnetic bead purification
AMPure XP beads	Beckman Coulter	A63881	Magnetic bead purification
Eppendorf 5424 Microcentrifuge	FisherScientific	05-403-93	centrifugation
INCU-Shaker 10 L	Benchmark Scientific	H1010	Cell culture
T100 Thermal Cycler	BIO RAD	1861096	Medium to High temperature cycling conditions
PTC-200 Thermal Cycler	GMI	8252-30-0001	Low temperature cycling conditions
RNase Away	Molecular Bioproducts	700S-11	Sterilization
50 mL Centrifuge Tube	Corning	430290	Nuclease-free
15 mL Centrifuge Tube	Corning	430052	Nuclease-free
Eppendorf tubes	USA Scientific	1615-5500	Nuclease-free
4200 Tapestation System	Agilent	G2991AA	Nucleotide analysis instrument for quality control of RNA and single stranded DNA samples
High Sensitivity RNA Screen Tape	Agilent	5067-5579	Quality control of RNA and single stranded DNA samples
RNA ScreenTape Sample Buffer	Agilent	5067-5577	Quality control of RNA and single stranded DNA samples
RNA ScreenTape Ladder	Agilent	5067-5578	Quality control of RNA and single stranded DNA samples
2100 Bioanalyzer Instrument	Agilent	G2939BA	Double stranded DNA quality control
High Sensitivity DNA Kit	Agilent	5067-4626	Quality control for double stranded cDNA samples
Water Bath	VWR	462-0244	Incubation
NanoDrop 2000/2000C Spectrophotometer	ThermoFisher	ND-2000C	Determination of RNA concentration