Probe‐Directed Degradation (PDD) for Flexible Removal of Unwanted cDNA Sequences from RNA‐Seq Libraries

Stuart K. Archer1, Nikolay E. Shirokikh1, Thomas Preiss2

1 These authors contributed equally to this work, 2 Victor Chang Cardiac Research Institute, Darlinghurst (Sydney), New South Wales
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 11.15
DOI:  10.1002/0471142905.hg1115s85
Online Posting Date:  April, 2015
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Abstract

Most applications for RNA‐seq require the depletion of abundant transcripts to gain greater coverage of the underlying transcriptome. The sequences to be targeted for depletion depend on application and species and in many cases may not be supported by commercial depletion kits. This unit describes a method for generating RNA‐seq libraries that incorporates probe‐directed degradation (PDD), which can deplete any unwanted sequence set, with the low‐bias split‐adapter method of library generation (although many other library generation methods are in principle compatible). The overall strategy is suitable for applications requiring customized sequence depletion or where faithful representation of fragment ends and lack of sequence bias is paramount. We provide guidelines to rapidly design specific probes against the target sequence, and a detailed protocol for library generation using the split‐adapter method including several strategies for streamlining the technique and reducing adapter dimer content. © 2015 by John Wiley & Sons, Inc.

Keywords: RNA‐seq; removal of rRNA; cDNA library; duplex‐specific nuclease; PDD

     
 
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Table of Contents

  • Introduction
  • Support Protocol 1: Design of Low‐Bias Degradation Deoxyribonucleotide Probes
  • Support Protocol 2: Starting Materials and Controlled Fragmentation of Total RNA
  • Alternate Protocol 1: Controlled Fragmentation of Total RNA with Escherichia coli RNase I
  • Support Protocol 3: End‐Repair and Polyadenylation of Fragmented RNA
  • Support Protocol 4: Synthesis of Complementary DNA using Split‐Adapter Primer
  • Support Protocol 5: Purification of cDNA Away from Excess Split‐Adapter Primer
  • Support Protocol 6: Circularization of cDNA using Intramolecular Ligation
  • Alternate Protocol 2: Circularization of cDNA in the Presence of Oligonucleotide Inhibiting Split‐Adapter self Ligation
  • Basic Protocol 1: Probe‐Directed Degradation of cDNA
  • Basic Protocol 2: Amplification and Size‐Selection of the PDD‐Treated cDNA
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Support Protocol 1: Design of Low‐Bias Degradation Deoxyribonucleotide Probes

  Materials
  • Computer with internet connection (command line instructions given are for the Bash Unix shell)
  • ApE (A Plasmid Editor, we use v2.0.47)
  • A working installation of Biopython (www.biopython.org)
  • Transcript sequences for your organism (refseq_rna database at NCBI and the target RNA sequence, from an established genome project)

Support Protocol 2: Starting Materials and Controlled Fragmentation of Total RNA

  Materials
  • NEBNext Magnesium RNA Fragmentation Module (NEB, cat. no. E6150)
  • 10× fragmentation buffer (NEBNext Magnesium RNA Fragmentation Module; NEB, cat. no. E6150)
  • Starting RNA (500 ng of total cellular RNA extracted from cells using standard procedures, such as cell lysis followed by phenol deproteinization, or kits such as TRIzol Plus RNA Purification System by Promega, RNeasy Mini Kit by QIAGEN, etc.)
  • Deionized nuclease‐free water
  • Iced water
  • 10× NEBNext RNA Fragmentation stop solution (NEBNext Magnesium RNA Fragmentation Module (NEB, cat. no. E6150)
  • 3 M NaOAc, pH 5.0
  • Glycogen (molecular biology grade), 10 mg/ml
  • 100% (v/v) ethanol
  • HE buffer (see recipe)
  • 80% ethanol, 20% (v/v) nuclease‐free deionized water
  • 200‐μl thin‐walled PCR tubes
  • Thermal cycler with heated lid
  • Vortex mixer
  • −20°C freezer
  • Centrifuge for 200‐μl and 1.5‐ml microcentrifuge tubes

Alternate Protocol 1: Controlled Fragmentation of Total RNA with Escherichia coli RNase I

  Materials
  • 0.5× cell lysate [about 100 mg; prepared, e.g., by grinding frozen suspension of 0.5× wet cell pellet in 0.5× HBB (w/v) with a cryomill]
  • Iced water
  • HEPES breakage buffer (HBB; see recipe)
  • E. coli RNase I (Thermo Scientific, cat. no. EN0601)
  • 1 M Tris·Cl, pH 2.0
  • 3 M NaOAc, pH 5.0
  • Stop solution (see recipe)
  • Deionized nuclease‐free water
  • 5:1 Phenol:chloroform mixture, pH 4.5
  • Absolute ethanol
  • HE or TE buffer (see reciperecipes)
  • 1.5‐ml microcentrifuge tubes
  • Vortex mixer
  • Microcentrifuge
  • NanoDrop ND‐1000 spectrophotometer or similar

Support Protocol 3: End‐Repair and Polyadenylation of Fragmented RNA

  Materials
  • Solution containing 500 ng of nucleic acids in 10 μl (see protocol 2)
  • Deionized nuclease‐free water
  • 10× T4 PNK Buffer (New England Biolabs, cat. no. B0201S) WITHOUT ATP
  • T4 Polynucleotide Kinase (T4 PNK; New England Biolabs, cat. no. M0201), NOT the T4 PNK 3′ phosphatase negative mutant
  • RNaseOUT RNase inhibitor (Life Technologies)
  • 10 mM EDTA
  • E‐PAP Buffer (Ambion/Life Technologies, cat. no. AM1350)
  • 25 mM MnCl 2 (Ambion/Life Technologies, cat. no. AM1350)
  • 50 mM rATP with neutral pH
  • 50 mM DTT (freshly made from 1 M stock)
  • 2 U/μl E‐PAP (Ambion/Life Technologies, cat. no. AM1350)
  • 3 M NaOAc, pH 5.0
  • Absolute ethanol
  • HE buffer (see recipe)
  • 1.5‐ml microcentifuge tubes
  • Thermostat for 1.5‐ml microcentrifuge tubes
  • Vortex mixer

Support Protocol 4: Synthesis of Complementary DNA using Split‐Adapter Primer

  Materials
  • Half (10 μl) of the 20 μl Polyadenylated RNA fragments solution (from step 18 of protocol 4)
  • 10 pmol/μl split‐adapter primer 5′ /5Phos/ GA TCG TCG GAC TGT AGA ACT CTG AAC G /iSp9/ G TGA CTG GAG TTC CTT GGC ACC CGA GAA TTC CAT TTT TTT TTT TTT TTT TTT TVN 3′ in nuclease‐free deionized water (iSp9: 9‐carbon spacer; 5Phos: 5 phosphorylation);
  • 10 mM dNTPs (Ambion/Life Technologies, cat. no. AM8200)
  • Iced water
  • 100 mM DTT (freshly made from 1 M stock)
  • 5× First‐Strand Buffer (Life Technologies, cat. no. 18080‐044)
  • 40 U/μl RNaseOUT RNase inhibitor (Life Technologies)
  • 200 U/μl SuperScript III reverse transcriptase (Life Technologies, cat. no. 18080‐044)
  • Deionized water
  • 200‐μl thin‐wall low‐binding nuclease‐free PCR microcentrifuge tubes
  • Sterile nuclease‐free 1.5‐ml microcentrifuge tubes
  • Thermal cycler with heated lid
NOTE: Primer sequence carries Illumina adapter sequences. Oligonucleotide sequences © 2007‐2013 Illumina, Inc. All rights reserved. Derivative works created by Illumina customers are authorized for use with Illumina instruments and products only. All other uses are strictly prohibited.

Support Protocol 5: Purification of cDNA Away from Excess Split‐Adapter Primer

  Materials
  • First‐strand cDNA solution (see protocol 5, step 9)
  • 20 U/μl E. coli Exo I (NEB, cat. no. M0293)
  • 500 mM EDTA, pH 8.0
  • Deionized nuclease‐free water
  • Agencourt AMPure XP purification system (Beckman Coulter, A63880)
  • PN binding buffer (see recipe)
  • PN washing buffer (see recipe)
  • 70% ethanol, 30% (v/v) deionized nuclease‐free water solution
  • HE buffer (see recipe)
  • RNase A/T1 Mix (Thermo Scientific, cat. no. EN0551)
  • Thermal cycler with heated lid
  • Vortex mixer
  • Low‐binding nuclease‐free 1.5‐ml microcentrifuge tubes (e.g., Eppendorf DNA LoBind tubes)
  • Two magnetic racks for 1.5‐ml microcentrifuge tubes
  • Heated oven to contain a magnetic rack set to 60°C
  • Heated block for 1.5‐ml microcentrifuge tubes set to 60°C
NOTE: If dealing with many samples, a 37°C heat block for 200‐μl tubes, or a second thermal cycler at 37°C, may be required at step 2 to facilitate batch operations.

Support Protocol 6: Circularization of cDNA using Intramolecular Ligation

  Materials
  • RNA‐free cDNA (∼250 ng/4 pmol) (see protocol 6, step 28)
  • Deionized nuclease‐free water
  • 10× CircLigase II Reaction Buffer (Epicentre/Illumina, cat. no. CL9021K)
  • 50 mM MnCl 2
  • 5 M betaine (Epicentre/Illumina, cat. no. CL9021K)
  • 100 U/μl CircLigase II (Epicentre/Illumina, cat. no. CL9021K)
  • 500 mM EDTA, pH 8.0
  • Agencourt AMPure XP purification system (Beckman Coulter, cat. no. A63880)
  • PN binding buffer (see recipe)
  • 70% ethanol, 30% (v/v) deionized nuclease‐free water solution
  • HE buffer (see recipe)
  • Low‐binding nuclease‐free thin‐walled 200‐μl PCR microcentrifuge tubes
  • Vortex mixer
  • Thermal cycler with heated lid
  • Low‐binding nuclease‐free 1.5‐ml microcentrifuge tubes
  • Magnetic rack for 1.5‐ml microcentrifuge tubes

Alternate Protocol 2: Circularization of cDNA in the Presence of Oligonucleotide Inhibiting Split‐Adapter self Ligation

  Additional Materials (also see protocol 6)
  • Annealing buffer (see recipe)
  • 10 pmol/μl blocking oligonucleotide 5′ TNB AAA AAA AAA AAA AAA AAA AAT GG /3BioTEG/ 3′ (ordered from IDT and dissolved in nuclease‐free deionized water)

Basic Protocol 1: Probe‐Directed Degradation of cDNA

  Materials
  • Solution containing circular ss cDNA (10 μl) (see protocol 7, step 22)
  • Stock mixture of all DSN probes at 2 μM each (ordered from IDT in a 96‐well plate format and dissolved at high individual concentrations in nuclease‐free deionized water, subsequently mixed together equally)
  • 10× DSN buffer (Evrogen)
  • Deionized nuclease‐free water
  • 1 U/μl DSN (Evrogen)
  • Iced water
  • 500 mM EDTA, pH 8.0
  • HE buffer (see recipe)
  • 25:24:1 Phenol:chloroform:isoamyl alcohol mixture, pH 8.0
  • Low‐binding nuclease‐free thin‐walled 200‐μl PCR microcentrifuge tubes
  • Vortex
  • Centrifuge
  • Low‐binding nuclease‐free 1.5‐ml microcentrifuge tubes
  • Thermal cycler with heated lid
  • Micro Bio‐Spin P‐6 columns (Bio‐Rad, cat. no. 732‐6221)

Basic Protocol 2: Amplification and Size‐Selection of the PDD‐Treated cDNA

  Materials
  • 12 μl of DSN‐treated circular ssDNA solution (see protocol 9, step 23)
  • Deionized nuclease‐free water
  • 10 mM dNTPs solution (each), PCR grade
  • 10× Pfx Buffer (Invitrogen/Life Technologies, cat. no. 11708‐013)
  • 50 mM MgSO 4 (Invitrogen/Life Technologies, cat. no. 11708‐013)
  • 10 μM forward primer: 5′ CAA GCA GAA GAC GGC ATA CGA GAT XXX XXX GTG ACT GGA GTT CCT TGG CAC CCG AGA ATT CCA 3′ (where XXX XXX is an Illumina indexing hexanucleotide sequence), ordered from IDT and dissolved in nuclease‐free deionized water.
  • 10 μM reverse primer: 5′ AAT GAT ACG GCG ACC ACC GAG ATC TAC ACG TTC AGA GTT CTA CAG TCC GA 3′ (ordered from IDT and dissolved in nuclease‐free deionized water)
  • 0.5 μg/μl Extreme Thermostable Single‐Stranded DNA Binding Protein (ET SSB protein; NEB, cat. no. M2401S)
  • 2.5 U/μl Platinum Pfx DNA Polymerase (Invitrogen/Life Technologies, cat. no. 11708‐013)
  • UltraPure Agarose (Invitrogen, cat. no. 16500‐500)
  • 0.5× TBE buffer (see recipe)
  • 6× GRGreen Loading Buffer (Excellgen, cat. no.‐EG‐1008)
  • 0.5 μg/μl 100 bp DNA Ladder (NEB, cat. no. N3231)
  • HE buffer (see recipe)
  • 3 M NaOAc, pH 8.0
  • 5 μg/μl nuclease‐free glycogen
  • Stop solution (see recipe)
  • 25:24:1 Phenol:chloroform:isoamyl alcohol mixture, pH 8.0
  • Absolute ethanol
  • 80% ethanol, 20% (v/v) deionized water solution
  • High Sensitivity DNA Kit (Agilent Technologies, cat. no. 5067‐4626)
  • Low‐binding thin‐walled nuclease‐free 200‐μl PCR microcentrifuge tubes
  • Vortex mixer
  • Thermal cycler with heated lid
  • 500‐ml Schott bottles
  • Microwave oven
  • Low‐binding nuclease‐free 1.5‐ml microcentrifuge tubes
  • Apparatus for horizontal electrophoresis allowing at least 100 × 7 (L×H) mm gels
  • Blue light transilluminator with optical filter
  • Sterile syringe with needle
  • Quantum Prep Freeze ‘N Squeeze DNA Gel Extraction Spin Columns (Bio‐Rad, cat. no. 732‐6165)
  • Thermoshaker for 1.5‐ml microcentrifuge tubes
  • 2100 Bioanalyzer (Agilent Technologies)
NOTE: Primer sequences are adapted from Illumina primer sequences.. All rights reserved. Derivative works created by Illumina customers are authorized for use with Illumina instruments and products only. All other uses are strictly prohibited.
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Literature Cited

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Key Reference
  Archer et al., 2014. See above.
  The original research article outlining the PDD method and analyzing its applicability for construction of RNA‐seq libraries.
Internet Resources
  http://github.com/stu2/blast2ape
  GitHub repository for Blast2Ape scripts.
  http://www.biomedcentral.com/content/supplementary/1471‐2164‐15‐401‐s3.zip
  Location of original paper supplementary files including alternative Perl‐based script.
  http://biologylabs.utah.edu/jorgensen/wayned/ape/
  ApE software.
  http://www.biopython.org
  Biopython software.
  http://www.ncbi.nlm.nih.gov/blast
  NCBI BLAST sequence homology search.
  http://primer3plus.com/cgi‐bin/dev/primer3plus.cgi
  Primer3 software.
  http://www.idtdna.com
  Web site of Integrated DNA Technologies (IDT) containing instruments for oligonucleotide ordering and some useful computational tools for nucleic acids analysis.
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