preparation of normalized cdna libraries for 454 sequencing

7/28/2019 Preparation of Normalized cDNA Libraries for 454 Sequencing

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Franois Pompanon and Aurlie Bonin (eds.), Data Production and Analysis in Population Genomics: Methods and Protocols,Methods in Molecular Biology, vol. 888, DOI 10.1007/978-1-61779-870-2_8, Springer Science+Business Media New York 2012

Chapter 8

Preparation of Normalized cDNA Libraries for 454 TitaniumTranscriptome Sequencing

Zhao Lai, Yi Zou, Nolan C. Kane, Jeong-Hyeon Choi, Xinguo Wang,and Loren H. Rieseberg

Abstract

Transcriptome sequencing from cDNA libraries has been extensively and efficiently used to analyzesequence variation in protein-coding genes (Expressed Sequence Tags) in eukaryote species. Rapidadvances in next-generation sequencing (NGS) technology, in terms of cost, speed, and throughput, allowus to address previously unanswerable questions in the fields of ecology, evolution, and systematics usingthese genomic tools. Transcriptome sequencing from individuals across different populations and speciesenables researchers to study the evolution of gene sequence variation at a population genomics level. Inthis chapter, we describe a customized protocol that has been successfully optimized for the developmentof normalized cDNA libraries in eukaryote systems suitable for Roche 454 GS FLX sequencing, requiringonly small quantities of starting material.

Key words: EST, Total RNA isolation, SMART cDNA synthesis, cDNA normalization, DSN, Roche454 GS FLX, Transcriptome sequencing

Expressed Sequence Tag (EST) sequencing of cDNA libraries hasbecome a powerful tool for dissecting the protein-coding fractionof a genome. Due to the considerable cost and time required for

deep sequencing of ESTs with Sanger methods, until very recentlyEST sequencing was limited to model species or key representa-tives of other taxa. In the past few years, however, with the rapiddevelopment of next-generation sequencing (NGS) techniquesand applications, whole transcriptome sequencing has beensuccessfully utilized as the basis for novel gene characterization,marker development, and population genomics studies in manynon-model species (e.g., ref. 1).

1. Introduction


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Among the currently available NGS platforms (e.g., Roche454, Illumina Genome Analyzer and HiSeq2000, Life SciencesSOLID), the Roche 454 Genome Sequencer FLX Titanium plat-form is particularly suitable for large-scale de novo EST sequencingin emerging and non-model organisms. 454 Titanium sequencing

can generate the long read lengths (approximately 400500 bp) inthe current NGS platforms, and the read lengths are comparable totraditional Sanger sequencing. Long reads are extremely useful forde novo transcriptome assembly for non-model species lacking

whole genome sequences or Sanger EST collections (e.g., ref. 2).Indeed, comparative analyses of the age distribution of duplicategenes based on Sanger, 454 Titanium, and Illumina sequence dataindicate that the shorter Illumina reads (paired end or not) fail toresolve close paralogs (3). In contrast, assemblies of Sanger and454 Titanium reads resolve similar proportions of close paralogs

(Ks < 0.1).454 EST sequencing requires generation of cDNA from RNA

samples. Due to the advantageous characteristic of largely full-length cDNA synthesis, SMART (Clontech) technology hasbecome the most widely adapted approach for cDNA synthesis.The method is based on synthesis of the first cDNA strand with ananchored oligo-dT using total RNA from eukaryote as template.The terminal C addition and template switching features of M-MLVreverse transcriptase, such as Superscript II Reverse Transcriptase,allow the synthesis of the first strand of cDNA (ss-cDNA) based on

full-length mRNA templates (see Fig. 1). Then, long-distancePCR is performed to amplify ss-cDNA to double-stranded cDNA(ds-cDNA) using high-fidelity DNA polymerase.

In order to maximize the rate of gene discovery, it is necessaryto employ a normalization strategy to remove high-abundancecDNA transcripts and generate a low-redundancy cDNA library.

We use a simple and efficient duplex-specific nuclease (DSN)normalization strategy using the TRIMMER cDNA normalizationkit (Evrogen), which is based on the denaturing and re-associationof cDNAs, followed by digestion with DSN. The enzymatic degra-

dation occurs primarily on the highly abundant cDNA fraction,and thus significantly and efficiently reduces the redundancy of acDNA library. The single-strand cDNA fraction is then amplifiedtwice by sequential PCR reactions according to the manufacturersprotocol.

However, owing to the nature of the pyrosequencing proce-dure adapted by Roche GS FLX 454, cDNA sequencing is espe-cially challenging for Roches 454 platform. Sequencing is sensitiveto homopolymers, such as the polyA/Ts existing in the sequences,

which not only result in excessive light production and cross talk

between neighboring cells, but also lead to polymerase delay andincomplete extension of template in the next cycle (4). We have,thus, developed a customized strategy to overcome homopolymer


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1218 Preparation of Normalized cDNA Libraries for 454 Titanium Transcriptome Sequencing

problems in 454 runs within cDNA samples. Firstly, we use amodified oligo-dT primer to prime the polyA+ tail of mRNAduring ss-cDNA synthesis (5). We further break down the polyA tailusing another modified oligo-dT primer during the ds-cDNA syn-thesis (5). These modified oligonucleotides are designed to effec-tively convert the long run of adenosine residues into a sequencethat causes fewer problems for pyrosequencing. Secondly, we

employ a different end polishing procedure so that only physicallyfragmented ends are polished (6). We design a unique adaptormodified from Illumina technology (7, 8) but using the 454 primer

A and primer B sequences. The ligation of cDNA fragments withadaptors results in the unique placement of ligation products(see Fig. 1). After PCR amplification, only those fragments withthe A primer at one end and the B primer at another end will beamplified. Lastly, because the tag sequence (M1 primer sequence,used for amplification of normalized cDNA) is in both ends, it ispossible to separate the internal fragments from the 5 and 3 end

fragments. The resulting library is a double-stranded DNA libraryinstead of the standard single-strand DNA library, making sizeselection much easier and more efficient (see Fig. 1).

Fig. 1. Overview of the procedures. See text for details.


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The overall workflow is described as follows (see Fig. 2). Firstly,total RNA is extracted from a biological source with the use ofTRIzolreagent and purified with the Qiagen RNeasy mini column,coupled with on-column DNase I treatment. Then, ss-cDNA syn-thesis is performed using SMART cDNA synthesis technique with

modified oligo dT primer to prime polyA+ of total RNA. ds-cDNAsynthesis converts ss-cDNA to ds-cDNA using PCR amplificationfollowing the Trimmer cDNA normalization manual. NormalizedcDNA is sonicated to 500800-bp fragments and the fragmentedends are polished and ligated with adaptors. The optimal ligationproducts are selectively amplified and subjected to two rounds ofsize selection, including gel electrophoresis and AMPure SPRI beadpurification. With this novel cDNA library preparation strategy, wehave successfully obtained approximately 250330 Mb total outputper half plate with a median read length of 480 bp, which is sub-

stantial improvement over standard ss-cDNA libraries.

Fig. 2. Overview of the workflow. The words in the boxesindicate the experiment steps and the text descriptions outside

of boxesindicate the experimental approaches.


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1. Misonix Ultrasonic Liquid Processors sonicator.

2. AMPure SPRI beads (Beckman).

3. 10 mM dNTPs.

4. Nuclease-free water.

5. T4 DNA polymerase (3 U/L, NEB).

6. Klenow fragment of DNA polymerase I (5 U/L, NEB).

7. Klenow Fragment (35 exo-) (50 U/L, NEB).

1. 10 mM dNTPs.

2. Nuclease-free water.

3. T4 DNA ligase (2,000 U/L, NEB).

4. Adaptor 454-1 (* indicates phosphorothioate bond, see

Note 5):5-CCATCTCATCCCTGCGTGTCTCCGACTCAGGCTCTTCCGATC*T-3.

5. Adaptor 454-2 (p indicates phosphate, see Note 5):

5 -pGATCGGAAGAGCCTGAGACTGCCAAGGCACACAGGGGATAGG-3.

6. Primer Ti-A: 5-CCATCTCATCCCTGCGTGTCTCCGACTCAG-3.

7. Primer Ti-B: 5-CCTATCCCCTGTGTGCCTTGGCAGTCT

CAG-3.8. Primer TiB-M1: 5-CCTATCCCCTGTGTGCCTTGGCAGT

CTCAGAAGCAGTGGTATCAACGCAGAGT-3.

1. GTG SeaKem agarose.

2. SYBR-safe dye.

3. 100 bp DNA ladder (NEB).

4. AMPure SPRI beads.

5. QIAquick Gel extraction kit (Qiagen).

6. Bioanalyzer DNA 7500 chip.

1. For plant tissues, we recommend using mortar and pestle togrind the tissues with liquid nitrogen. Add 1 mL ofTRIzolReagent directly to the tube containing the frozen tissue

powder (approximately 500

L). Vortex mixture thoroughlyto homogenize tissues. Incubate mixture for 5 min at roomtemperature.

2.4. cDNA

Fragmentation, End

Polishing, Adding

A to 3 End

2.5. Ligation

with Adaptors

and Amplification

2.6. Final Size

Selection and Library

Quantification

3. Methods

3.1. Total RNA

Extraction and

Purification


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2. Add 1/5 volume (200 L) of chloroform to each sample.Immediately shake the mixture vigorously for 15 s. Centrifugethe mixture for 15 min at 4C at 12,000 g.

3. Transfer upper aqueous phase (approximately 600 L) tolabeled RNase-free 1.7-mL tube. Precipitate RNA by adding0.53 volume (approximately 320 L) of 100% ethanol. Gentlypipette to mix.

4. Transfer mixture, including any precipitate that may haveformed, to Qiagen RNeasy mini-spin column. Proceed immedi-ately. Follow manufacturers instructions in the next few steps.

5. Centrifuge for 30 s at 12,000 g. Discard flow through.

6. On-Column DNase Treatment: Wash the column with 350 Lof Buffer RW1. Spin for 30 s. Discard the flow through. Pipettethe DNase I/RDD Buffer mixture 80 L (add 70 L of RDD

Buffer to 10 L of DNase I stock solution; gently mix) directlyonto the RNeasy silica-gel membrane of the RNeasy mini-spincolumn. Incubate for 15 min at room temperature. Wash thecolumn with another 350 L of Buffer RW1. Spin for 30 s.Discard the flow through.

7. Wash the column twice with 500 L of Buffer RPE each. Spinfor 30 s. Discard flow through.

8. Centrifuge for 1 min to dry the column. Transfer column to anew 1.5-mL tube.

9. Apply 30 L of RNase-free water directly onto silica-gel mem-brane. Incubate for 1 min at room temperature. Spin for 1 min.

10. The elution now contains purified total RNA. Determine RNAconcentration with NanoDrop. Determine integrity usingBioanalyzer/RNA 6000 Nano kit.

1. Prepare total RNA/primer mixture in a 0.2-mL PCR tube.The components per reaction are as follows: 1 L CDSIII-1Mprimer, 11.5 g total RNA (maximum volume of 3 L), and1 l SMART IIA primer. Add DNase and RNase-free water to

bring a final volume of 5 L. Mix gently.

2. Incubate for 3 min at 65C and quick chill for 2 min on ice.Spin briefly. Keep on ice.

3. At room temperature, prepare first-strand master mix. Thecomponent per reaction is as follows: 2 L 5 First-StrandBuffer, 1 L 10 mM dNTPs, 1 L 100 mM DTT, and 1 LSuperScript II. Gently pipette to mix. Spin briefly.

4. Incubate the reaction in PCR thermo-cycler for 1 h at 42C.Then, inactivate the enzyme activity by incubating the reaction

for 15 min at 70C.5. Remove from thermo-cycler. Spin briefly. Place samples on ice.

Immediately proceed to ds-cDNA synthesis.

3.2. First-Strand

and Double-Stranded

cDNA Synthesis

3.2.1. First-Strand cDNASynthesis


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1. Prepare second-strand master mix on ice. The components perreaction are as follows: 69.5 L Nuclease-free water, 20 L 5HF PCR Buffer, 2 L 10 mM dNTP Mix, 2 L SMARTIIA_DNA primer, 2 L CDSIII-2M primer, 1.5 L MgCl

2,

and 1 L Phusion Hot Start DNA Polymerase. Usually, two

reactions per sample are required.

2. Add 2 L ss-cDNA template to each tube. Add 98 L second-strand master mix to make 100-L total volume. Mix gently.Centrifuge briefly.

3. PCR program: 98C for 1 min, and then 98C for 7 s; 66Cfor 20 s; and 72C for 5 min for 1820 cycles.

4. When the PCR cycling program is completed, check 5 L ofeach PCR product alongside the 1 kb DNA ladder on regular1% TBE agarose gel. A moderately strong smear of PCR

amplification ranging from 500 bp to 4 kb indicates the goodexample of ds-cDNA for most plant species.

5. Purify the amplified ds-cDNA using the Qiagen PCRpurification Kit following the manufacturers instructions. Thefinal elution volume is 20 L EB buffer.

6. Ascertain the concentration of purified ds-cDNA by Nanodrop.800 ng cDNA are needed for the normalization step. If theresulting concentration is less than 1 g in 20 L, concentrateusing a Microcon YM-30 column.

1. The following procedures are based on the Evrogen TrimmercDNA normalization kit manual with a few modifications.

2. Prepare the cDNA hybridization step. The components perreaction for three reactions are as follows: 800 g (9 L)cDNA from the previous step, 3 L 4 hybridization buffer,and add H

2O to a final volume of 12 L.

3. Aliquot 4 L cDNA hybridization mix into three individualPCR tubes and overlay each with a drop of sterile mineral oil;centrifuge briefly to collect liquid and separate phases.

4. Incubate the tubes at 98C for 2 min in a thermal-cycler todenature the ds-cDNA, and then at 68C for 5 h. Proceedimmediately to the next step.

5. Dilution of the DSN and checking of the DSN activity beforestarting a new tube are based on the manual.

6. DSN treatment: Near the end of the hybridization period, pre-heat the DSN master buffer at 68C for 5 min. Prepare and strength dilutions of the DSN using DSN storage buffer asthe diluents; store on ice until ready to use. At the end of the

hybridization period, add 5 L preheated master buffer to eachtube. Spin briefly in a benchtop centrifuge 2 and return imme-diately to the thermal-cycler. To three individual PCR tubes,

3.2.2. Double-Stranded

cDNA Synthesis

3.3. cDNA

Normalization

and Amplification

3.3.1. cDNA Normalization


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add 1 L dilution DSN enzyme, 1 L dilution DSNenzyme, and 1 L DSN storage buffer (as control) separately.Incubate at 68C for 25 min. Add 10 L of DSN stop solutionto each tube, mix well, and spin briefly. Add 20 L H

2O to each

tube and then store at 20C or proceed with the next step.

1. Prepare the master mix for first amplification of normalizedcDNA: Set up three separate PCR reactions for each reactionabove; components per reaction are as follows: 34.85 L H

2O,

10 L 5 HF PCR buffer, 1 L 10 mM dNTPs, 1.5 L PrimerM1 (10 M), 0.75 L 50 mM MgCl

2, 0.4 L Phusion

Hot start Polymerase, and 1.5 L Normalized cDNA fromabove. Amplify using the following thermal profile: 98C for1 min; 98C for 7 s, 66C for 20 s, and 72C for 4 min forseven cycles.

2. Take out all tubes from thermal-cycler. Put the experimentaltubes ( and DSN-treated tubes) on ice. Remove a 5-Laliquot from the control tube and set this aside.

3. Amplify the control tube (0 DSN, just storage buffer tube)for an additional two cycles (total = 9). Remove another 5-Laliquot and set this aside.

4. Repeat step 3 twice more, producing aliquots from this tubethat correspond to 11 and 13 cycles.

5. Load 5-L aliquots from each 7th-, 9th-, 11th-, and 13th-

cycle PCR products side by side on an agarose gel to evaluateoptimum cycle number Xas described in the manufacturersinstructions, where X= optimal numbers of cycles required foramplification of each of the control tube (see Note 6).

6. Return experimental tubes to the thermal-cycler and amplifyfor an additional N + Xcycles, where N=X 7 (see Note 6).

7. Load 5 L on a gel to determine which enzyme dilutiontreatment ( or ) gives the best results, as described in themanual (see Note 7).

8. Once optimum enzyme treatment has been established, makedilutions of first amplified normalized cDNAs by adding 5-LcDNA aliquots into 50 L H

2O. This will be used as the

template for the second amplification of normalized cDNA.

1. Set up two separate 100 L PCR reactions for each sample.The components per reaction are as follows: 69.7 L H

2O,

20 L 5 HF PCR buffer, 2 L 10 mM dNTPs, 4 L PrimerM2 (10 M), 1.5 L 50 mM MgCl

2, 0.8 L Phusion Hot

start Polymerase, and 2 L first amplified diluted cDNA

from above.2. Amplify using the following PCR program: 98C for 1 min;

98C for 7 s, 64C for 20 s, and 72C for 4 min for 12 cycles.

3.3.2. First Amplification

of Normalized cDNA

3.3.3. Second Amplification

of Normalized cDNA


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3. Pool the products, purify on a Qiagen Qiaquick column, elutein 50 L EB buffer, and quantify them using a Nanodrop.Normalized cDNA can be stored at 20C.

1. For each sample, dilute 2.02.5 g second amplified cDNA

from above into EB buffer for a final volume of 100 L.

2. Load the diluted samples into the Misonix Ultrasonic LiquidProcessors sonicator, ensuring that the liquid level in the tubesis below the liquid level in the sonicator. Ensure that thetubes are equally spaced around the center of the cup horn,and at equal depths in the water. Close the latch on the soni-cator door.

3. Sonicate the tubes at the following intervals at amplitude of 1.Use the following program for the sonicator: 30-s cycles, 30-s

rest time, and 40-sprocessing time.4. Check the result of sonication by running 2.5 L of each of

sonicated cDNA on regular 1% TBE agarose gel. The targetsize of fragments ranges from 300 to 1,000 bp.

1. This process is performed following the AMPure beadsmanual. Note that each bottle of AMPure beads has to becalibrated before starting the new bottle. The target sizeremoval is the fragments less than 500 bp. So use the ratio ofbeads that will remove fragments less than 500 bp (usually

0.5 or 0.55).2. Measure the volume of the samples using the pipettor. Add EB

Buffer to a final volume of 100 L. Add the needed amount ofAMPure beads (volume is relative to the amount of startingsonicated cDNA, usually will be 50 or 55 L). Vortex for1020 s followed by a quick centrifuge. Incubate for 5 min atroom temperature.

3. Using a Magnetic Particle Collector (MPC), pellet the beadsagainst the wall of the tube. Let the tubes stand in the MPC for2 or 3 min.

4. Remove supernatant with a pipette carefully. Wash beads twicewith 500 L of 70% ethanol. Incubate each wash at roomtemperature for 30 s before removing with a pipette.

5. Remove all the supernatant carefully. To dry completely, placeon a preheated 4550C heating block for about 5 min.

6. Add 20 L EB buffer to each tube. Vortex each tube for1020 s and then centrifuge it briefly. This step elutes thecDNA from the AMPure beads.

7. Using the MPC, pellet the beads against the wall of the tubeonce more, and transfer the supernatant containing thepurified, sonicated cDNA to a new tube. Quantify cDNA

3.4. cDNA

Fragmentation, End

Polishing, Adding

A to 3 End

3.4.1. cDNA Fragmentation

by Sonication

3.4.2. Small Fragment

Removal


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concentration using a Nanodrop. If the amount of cDNA isgreater than 200 ng, proceed to the next step (see Note 8).

1. Polish the fragmented cDNA to ensure that all ends areblunted. Prepare the end-polishing reaction master mix by

combining the following in a tube at room temperature: 20 Lwater, 5 L 10 NEB2 buffer, 5 L 10 BSA, 2 L 10 mMdNTPs, 1.4 L T4 DNA polymerase (4 U), and 0.8 L Klenowfragment of DNA polymerase I (4 U). Aliquot 34.2 L mastermix into each sample tube. Add 200500 ng fragmentedcDNA from the previous step. Mix gently (see Note 9).

2. Incubate the reaction at room temperature for one and a halfhours, and then heat to inactivate the enzyme activity at 70Cfor 15 min.

3. The reaction products are purified by Qiaquick column.Elution volume is 32 L.

1. Prepare the adding A master mix: 2.7 L water, 5 L 10NEB2 buffer, 10 L 1 mM dATP, and 0.3 L Klenow 35exo (15 U). Aliquot 18 L master mix into each sample tube.

Add 32 L end-polished cDNA from the previous step to makethe reaction total volume of 50 L. Mix gently.

2. Incubate the reaction at 37C for 30 min.

3. The reaction products are purified by Qiaquick column. Elute

with 30 L EB buffer.

1. Prepare the ligation master mix: 13.6 L water, 5 L T4 DNAligase buffer, 1 L of mix adaptor of 454-1 and 454-2, and0.4 L T4 ligase. Add 20 L master mix into each sample tube.

Add 30 L 3 A-added cDNA from the previous step to makethe reaction total volume of 50 L. Mix gently.

2. Incubate the reaction at 16C for 30 min.

3. The reaction products are purified by Qiaquick column. Elute

with 30 L EB buffer.

1. Set up four PCR test tubes for each sample and label them as1, 2, 3, and 4. Add 1 L ligation product from the previousstep into each tube. Add 2 L Ti-A primer into the tube 1,2 L Ti-B primer into the tube 2, 1 L Ti-A primer and 1 LTi-B primer into the tube 3, and 1 L Ti-A primer and 1 LTi-B-M1 primer into the tube 4.

2. Prepare the PCR master mix. Assemble the component perreaction as follows: 34.75 L water, 10 L 5 HF PCR buffer,

1 L 10 mM dNTPs, 0.75 L 50 mM MgCl2, and 0.5 LPhusion Hot Start Polymerase. Aliquot 47 L master mixinto each sample tube. Gently mix.

3.4.3. cDNA End Polishing

3.4.4. Adding A to 3 End

3.5. Ligation

with Adaptors

and Amplification

3.5.1. cDNA Ligation

with Adaptors

3.5.2. PCR Test of Ligation


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3. Amplify using the following PCR program: 98C for 30 s;and then 98C for 7 s, 65C for 30 s, and 72C for 1 min for16 cycles.

4. Check the result of the PCR test by running 5 L of each PCRproduct on an agarose gel. The expected results are no prod-ucts from the tube 1 and 2 and a nice smear of PCR productsranging from 500 to 1,000 bp from tubes 3 and 4.

5. If the PCR test result is as expected, then proceed to the nextstep for final library amplification.

1. Set up two PCR tubes for each sample, and label them as 1 and2. Add 2 L ligation product from the previous step intoeach tube. Add 2 L Ti-A primer and 2 L Ti-B primer intothe tube 1 for amplifying cDNA internal fragments. Add 2 L

Ti-A primer and 2 L Ti-B-M1 primer into the tube 2 foramplifying 5 end and 3 end of cDNA fragments (see Note 10).

2. Prepare the final library amplification PCR master mix.Assemble the component per reaction as follows: 69.5 Lwater, 20 L 5 HF PCR buffer, 2 L 10 mM dNTPs, 1.5 L50 mM MgCl

2, and 1 L Phusion Hot Start Polymerase.

Aliquot 94 L master mix into each sample tube. Mix gently.

3. Amplify using the following PCR program: 98C for 30 s; 98Cfor 7 s, 65C for 30 s, and 72C for 1 min for 16 cycles.

4. The reaction products are purified by Qiaquick column. Elutewith 14 L EB buffer. Quantify the purified products using aNanodrop.

5. If the total cDNA amount of tube 1 and tube 2 from eachsample is in the range of 36 g, then proceed to the next stepfor final size selection.

1. Prepare a 0.8% agarose gel using SeaKem GTG agarose/1TAE buffer with SYBR-SAFE Dye.

2. Prepare samples for gel loading. For each sample, mix up to

24 L samples (36 g of cDNA) with 8 L loading dye.Prepare one aliquot of ladder per sample, according to thefollowing: 5 L water, 3 L Tit-gel Dye, and 2 L 100 bpDNA Ladder.

3. Loading samples: Leave at least one well between each ladderand sample, placing a ladder before each sample. Run the gelat 100 V for 2 h.

4. Place the gel on the DarkReader. Carefully cut the PCR prod-ucts ranging from 500 to 800 bp. Put the gel slice into a 2-mLtube.

5. Isolate the cDNA from the gel slice according to the QiagenQiaquick Gel Isolation Kit instructions for the next few steps.

3.5.3. Final Library

Amplification

3.6. Final Size

Selection and Library

Quantification


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6. Add 3 volume of Buffer QG to the tube with gel slice. Putinto the turboMix to shake for 5 min until the gel dissolvescompletely.

7. Add 1 volume of isopropanol and mix. Apply the sample toQIAquick column and spin for 1 min. Discard the flow through.

Add 500 L Buffer QG to the column and spin for 1 min.8. Wash the column twice with 500 L PE buffer. Incubate PE

buffer for 25 min before spinning. Discard the flow through.Spin for additional 1 min to dry the column.

9. Elute the column twice with 25 L EB buffer each.

10. Add 25 L (0.5) AMPure SPRI beads to the eluted sample.This step will remove any fragments less than 500 bp. Wash thebeads with 12 L EB buffer.

11. Assess the size distribution of the completed library using

Bioanalyzer with an Agilent DNA 7500 chip (see Fig. 3).12. Quantify the final library concentration using Quan-It

PicoGreen.

1. It is assumed that users have knowledge of molecular biologytechniques and safe laboratory practices. Before undertaking a

new protocol or using unfamiliar reagents, users should reviewrelevant Material Safety Data Sheets to identify potential hazardsand recommended precautions. For background in generalmolecular biology, please see ref. 9.

4. Notes

Fig. 3. Electrophrogram of the final cDNA library assessed by a Bioanalyzer DNA7500 chip. The electrophrogram shows

the high quality of the final library with the average size of 816 bp and no noise before 500 bp and after 1,000 bp.


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132 Z. Lai et al.

2. For all steps involved in RNA isolation, it is important to guardagainst sources of dust and nucleases. All reagents must beprepared with RNase-free water using RNase-free chemicalsand DNase- and RNase-free plastics. In addition, decontami-nate both workspaces and pipettes with RNase Zap, according

to manufacturers instructions. Among the reagents used inthe whole process, TRIzol contains phenol, which is toxic incontact with skin and if swallowed. Thus, it should be handled

with caution and should therefore be manipulated under afume hood using gloves.

3. SMART IIA primer is used for ss-cDNA synthesis. This is theRNA-based primer and the last three Gs need to be RNA basesif you synthesize your own. Therefore, this primer needs to bediluted with RNase-free water, aliquoted, and stored at 80C.

4. SMART IIA_DNA primer is used for ds-cDNA synthesis. It isused together with CDSIII-2M primer to further break downthe polyA/T homopolymer sequences. This primer is a regularDNA-based primer.

5. The design of the Adaptor 454-1 and Adaptor 454-2 used forthe ligation is based on the Illumina technology. So when syn-thesizing your own adaptors, a phosphorothioate bond isneeded for the Adaptor 454-1 and a 5 end phosphate is neededfor the Adaptor 454-2. These adaptors are modified for further454 sequencing and thus are based on the 454 primer A and

primer B sequences. The part of 454 sequence from Adaptor454-1 is in the same orientation as the original 454 primer A:5-CCATCTCATCCCTGCGTGTCTCCGACTCAG-3 . Thepart of 454 sequence from Adaptor 454-2 is the reverse-complementary sequence of the original 454 primer B:5-CTGAGACTGCCAAGGCACACAGGGGATAGG-3 .Prepare the mix of adaptor by mixing the primers at the finalconcentration of 10 M each. All primers should be purified byhigh-performance liquid chromatography (HPLC) grade.

6. For each sample, determine optimal cycle number, X. The

optimal cycle should show a fairly dark smear in the 500 bp to4 kb range, and no nonspecific amplification above the 8 kbrange. In most of our cases, PCR product from 9th cyclescontrol tube yielded the optimum results, so X= 9. Then, thenumber of additional cycles to run is N+X, where N=X 7.For instance, ifX= 9, N= 9 7 = 2. Then, 2 + 9 = 11; more cyclesare needed for these 7-cycle experimental tubes and 18 cyclesin total for experimental tubes.

7. During the DSN normalization step, most of the time, boththe and DNS dilution tubes contain good amplificationproducts and can be combined for the next cDNA fragmenta-tion step.


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8. Small fragment removal using AMPure beads is performedtwice in the protocol. The removal of the small fragments aftercDNA fragmentation ensures that fragments of the right sizeare ligated to the adaptor. If small fragments remain in the finalcDNA library, they will be preferably amplified during the

following emPCR amplification and this will result in shortsequence reads.

9. For the end-polishing step, it is important that no kinase isadded (7) so that the new ends resulting from the frag-mentation/polishing will have 5 phosphates to ligate withthe adaptor. Original 5 end and 3 end with the tag sequence(corresponding to the M1 sequence) will not ligate with theadaptor because they do not bear 5 phosphates.

10. With a separate PCR amplification of ligation products, cDNA

internal fragments and end fragments are generated. The primerA sequence, which corresponds to the 454 Titanium sequencingprimer, will always correspond to internal cDNA sites, thus mini-mizing the chance of hitting polyA/T homopolymer sequences.

Acknowledgments

The authors thank James Ford, Jade Buchanan-Carter, Zach Smith,

and Keithanne Mockaitis for technical support for sequencing, andJie Huang for assistance with manuscript preparation. This workwas supported in part by a Roche Applied Science/454 LifeSciences 1 GB sequencing grant program to Z.L. and L.H.R., aNatural Sciences and Engineering Research Council of Canada(NSERC) grant #353026 to L.H.R, and the Indiana METACytInitiative of Indiana University, funded in part through a majorgrant from the Lilly Endowment, Inc.

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