... sequencing is the amount of time and resources required for template and library preparation. 1. ... Autoclave and let it cool to room temperature before use. 2.
Chapter 3 Construction of RNA-Seq Libraries from Large and Microscopic Tissues for the Illumina Sequencing Platform Hagop S. Atamian and Isgouhi Kaloshian Abstract Second-generation DNA sequencing platforms have emerged as powerful tools in biological research. Their high sequence output at lower cost and minimal input DNA requirement render them suitable for broad applications ranging from gene expression studies to personalized clinical diagnostics. Here, we describe the preparation of cDNA libraries, from both whole aphid insects and their microscopic salivary gland tissues, suitable for high-throughput DNA sequencing on the Illumina platform. Key words: Second-generation sequencing, RNA from minute amount of tissue, Illumina, cDNA synthesis
1. Introduction Classical DNA sequencing, based on the Sanger method, improved the diagnosis of diseases and contributed to our understanding of the various biological processes, identification of new drug targets, and classification of organisms. The launch of new sequencing technology referred to as “second-generation” sequencing made possible the incorporation of sequencing in even more studies aimed at finding answers to numerous biological questions, something which would not have been economically and rationally practical before (1). Second-generation sequencing technology is currently commercially available on five major platforms: AbI3730xl Genome Analyzer, Roche (454), Illumina Genome Analyzer, ABISOLiD, and HeliScope (2). Regardless of the platform, one of the bottlenecks for next-generation sequencing is the amount of time and resources required for template and library preparation.
Hailing Jin and Walter Gassmann (eds.), RNA Abundance Analysis: Methods and Protocols, Methods in Molecular Biology, vol. 883, DOI 10.1007/978-1-61779-839-9_3, © Springer Science+Business Media, LLC 2012
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Here, we describe the stepwise preparation of a cDNA library (also referred to as an mRNA-Seq library) suitable for sequencing with the Illumina Genome Analyzer platform. Using this protocol, we prepared high-quality mRNA-Seq libraries from whole aphids as well as microdissected aphid salivary gland tissues.
2. Materials Prepare all solutions using diethyl pyrocarbonate (DEPC)-treated water to prevent RNA degradation by RNase enzymes. To further minimize RNA degradation, keep samples on ice at all times, unless indicated otherwise. Wear gloves and use sterile techniques when working with RNA. First-time RNA users are encouraged to read “General Remarks on Handling RNA.” All glassware and plasticware should be RNase free. All centrifugation steps are performed at 4°C, unless indicated otherwise. 1. DEPC-treated water: Add 1 ml of 0.1% DEPC to 1,000 ml distilled water (see Note 1). Mix well on a magnetic stirrer overnight at room temperature until completely dissolved. Autoclave and let it cool to room temperature before use. 2. TRIzol (Invitrogen). 3. Chloroform. 4. 100% Isopropanol. 5. Nucleic acid carrier (linear polyacrylamide). 6. 70% Ethanol (EtOH), diluted with RNase-free DEPC-treated water. 7. RNeasy kit (containing RLT, RW1, and RPE Buffers; QIAGEN). 8. QIAshredder (QIAGEN). 9. DNaseI enzyme and DNaseI buffer. 10. Phenol. 11. 3 M sodium acetate (NaOAc), pH 5.2. 12. 100% EtOH. 13. mRNA-Seq Sample Prep Kit (containing Sera-mag oligo(dT) beads, Bead Binding Buffer, Bead Washing Buffer: 10 mM Tris–HCl, 5× Fragmentation Buffer, Fragmentation Stop Solution, Glycogen, Random Primers, 5× First-Strand Buffer, 25 mM dNTP, RNaseOUT, GEX Second Strand Buffer, RNaseH, DNA Pol I, 10× End Repair Buffer, T4 DNA Polymerase, Klenow DNA Polymerase, T4 Polynucleotide Kinase, 10× A-Tailing Buffer, 1 mM dATP, Klenow Exoenzyme, 2× Rapid T4 DNA Ligase Buffer, 1.5 μM PE Adapter Oligo Mix, T4 DNA Ligase, 5× Phusion Buffer, 25 μM PCR
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Primer PE 1.0, 25 μM PCR Primer PE 2.0, Phusion DNA Polymerase; Illumina). 14. 100 mM DTT. 15. SuperScript III Reverse Transcriptase (Invitrogen). 16. Elution buffer: 10 mM Tris–HCl, pH 8.5. 17. PCR Purification Kit (QIAGEN). 18. MinElute PCR Purification Kit (QIAGEN). 19. Agarose: Regular and high resolution for separation of small nucleic acids. 20. 1× Tris acetate–EDTA (TAE) buffer: 40 mM Tris acetate and 1 mM EDTA. 21. Loading dye. 22. 100 bp DNA ladder. 23. Gel Extraction Kit (QIAGEN). 24. GeneCatcher Disposable Gel Excision Kit (The Gel Company).
3. Methods 3.1. Total RNA Extraction from Aphid Salivary Gland Tissue
1. Collect microdissected salivary gland tissue in a 1.5-ml Eppendorf tube containing 100 μl of TRIzol reagent on ice. 2. Homogenize the tissue thoroughly with a small pestle for 2 min at room temperature (see Note 2). Rinse the pestle with an additional 100 μl of TRIzol reagent into the tube to remove excess tissue and proceed quickly to the next step. The tube can be frozen immediately in liquid nitrogen and stored at −80°C until use. 3. Vortex the tube thoroughly and incubate for 5 min at room temperature to allow for the complete dissociation of nucleoprotein complexes. 4. Add 40 μl of chloroform (1/5th the volume of TRIzol) and vortex vigorously for 20 s. 5. Incubate the tube at room temperature for 5 min. Centrifuge at 12,000 × g for 15 min. 6. Carefully transfer the supernatant to a fresh tube (see Note 3). Add equal volume of isopropanol and 25 μg of a carrier for nucleic acids, such as linear polyacrylamide, to maximize recovery. 7. Mix by inverting (DO NOT VORTEX), spin briefly (1–2 s), and store the tube at -20°C overnight for RNA precipitation. 8. Centrifuge the tube at 12,000 × g for 10 min. Carefully pipette out the liquid (see Note 4).
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9. Wash the pellet with 1 ml of 70% EtOH, invert the tube three to four times, and centrifuge at 7,500 × g for 10 min. 10. Pipette out the EtOH without disturbing the pellet (see Note 5). Let the tube dry on ice for 10 min and resuspend the pellet in 10–15 μl of RNase-free DEPC-treated water for 30 min. 3.2. Total RNA Extraction from Whole Aphids
1. For RNA isolation from large amount of tissue such as whole aphids, the RNeasy kit is used and steps 2–11 are carried out at room temperature, including centrifugation. 2. Grind 40 adult aphids to a fine powder in a 1.5-ml Eppendorf tube, submerged in liquid nitrogen, using a small pestle attached to an electric drill (see Note 2). 3. Transfer 100 mg of the powder to a 2-ml Eppendorf tube containing 1 ml of the RLT Buffer. 4. Homogenize the mixture by passing ten times through an 18-gauge needle. Add an additional 1 ml of the RLT Buffer to the homogenate. 5. Pass the homogenate through the QIAshredder by centrifuging at 4,000 × g for 2 min. Transfer the supernatant to a clean tube. 6. Add one volume of 70% EtOH to the homogenized lysate and mix immediately by shaking vigorously. 7. Immediately apply the sample to an RNeasy midi column. Centrifuge at 4,000 × g for 5 min and discard the flow through. 8. Add 4 ml of the RW1 Buffer to the RNeasy column and centrifuge at 4,000 × g for 5 min to wash the column. Discard the flow through. 9. Add 2.5 ml of the RPE Buffer to the RNeasy column and centrifuge at 4,000 × g for 2 min. Discard the flow through. 10. Add another 2.5 ml of the RPE Buffer to the RNeasy column and centrifuge at 4,000 × g for 5 min. Discard the flow through. This helps dry the RNeasy silica-gel membrane. 11. Add 150–250 μl of RNase-free DEPC-treated water to elute the RNA. Incubate for 1 min and centrifuge at 4,000 × g for 3 min.
3.3. DNase Treatment
1. Add 20 μl of DNaseI Buffer and 4 U of DNaseI enzyme to 20 μg of RNA and adjust the final volume to 200 μl with RNase-free DEPC-treated water. 2. Incubate the tube at 37°C for 30 min. 3. Add 300 μl of RNase-free DEPC-treated water to bring the final volume to 500 μl. Add 1/2 volume of phenol and 1/2 volume of chloroform. Vortex vigorously.
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4. Centrifuge at 12,000 × g for 15 min. 5. Transfer the supernatant to a fresh tube. Add 1/10 volume of 3 M NaOAc, two volumes of 100% EtOH, and 25 μg of the nucleic acid carrier. 6. Incubate the tube at −20°C for at least 2 h. 7. Centrifuge at 18,000 × g for 30 min. 8. Carefully pipette out the liquid (see Note 4). 9. Wash the pellet with 1 ml of 70% EtOH and invert the tube three to four times. Incubate the tube on ice for 5 min before centrifugation to dissolve all the salts. 10. Centrifuge at 12,000 × g for 5 min. 11. Pipette out the EtOH without disturbing the pellet (see Note 5). Air dry the pellet on ice for 10 min and resuspend in 10–15 μl of RNase-free DEPC-treated water for 30 min on ice. 12. Amplify a housekeeping gene with PCR using 1 μl of the DNaseI-treated RNA as template to ascertain that there is no DNA contamination (see Note 6). 13. Check the quality and the concentration of the DNase-free RNA using instruments that measure low quantities of nucleic acids, such as an Agilent 2100 Bioanalyzer (Agilent Technologies) (see Note 7). Store the RNA at −80°C. 3.4. mRNA Purification from Total RNA
The reagents used in the steps below are provided either by the Ilumina’s mRNA-Seq Sample Prep Kit or listed in materials. Illumina recommends to start with 1–10 μg of total RNA. We have constructed libraries from 100 ng and 4 or 7 μg of total RNA as starting material. The recommended quantities of the reagents are for 1–10 μg of total RNA. Use half the amount of the recommended reagents for libraries with starting material of
