制备植物复用小型RNA库

Kerrigan B. Gilbert; Noah Fahlgren; Kristin D. Kasschau; Elisabeth J. Chapman; James C. Carrington; Alberto Carbonell

doi:10.21769/BioProtoc.1275

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Peer-reviewed

Preparation of Multiplexed Small RNA Libraries from Plants

制备植物复用小型RNA库

KG Kerrigan B. Gilbert

NF Noah Fahlgren

KK Kristin D. Kasschau

EC Elisabeth J. Chapman

JC James C. Carrington

Alberto Carbonell email

发布: 2014年11月05日第4卷第21期 DOI: 10.21769/BioProtoc.1275 浏览次数: 13401

评审: Tie LiuFeng Li

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参见作者原研究论文

The authors used this protocol in:

Cover of Plant Physiology, featuring study using the protocol.

May 2014

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Abstract

High-throughput sequencing is a powerful tool for exploring small RNA populations in plants. The ever-increasing output from an Illumina Sequencing System allows for multiplexing multiple samples while still obtaining sufficient data for small RNA discovery and characterization. Here we describe a protocol for generating multiplexed small RNA libraries for sequencing up to 12 samples in one lane of an Illumina HiSeq System single-end, 50 base pair run. RNA ligases are used to add the 3’ and 5’ adaptors to purified small RNAs; ligation products that lack a small RNA molecule (adaptor-adaptor products) are intentionally depleted. After cDNA synthesis, a linear PCR step amplifies the DNA fragments. The 3’ PCR primers used here include unique 6-nucleotide sequences to allow for multiplexing up to 12 samples.

Keywords: Small RNA (小核糖核酸)

High-throughput sequencing (高通量测序技术)

Library preparation (文库制备)

Multiplexing (复用)

SRNA-seq (sRNA序列)

Materials and Reagents

Low molecular weight gel-based small RNA isolation
1. ssRNA marker (Takara Bio Company, catalog number: 3416 )
2. Reagents for 17% polyacrylamide, 7M Urea PAGE
  1. 37.5:1 polyacrylamide:bisacrylamide (see Recipes)
  2. Urea (Life Technologies, catalog number: 15505-050 )
  3. TEMED (Life Technologies, catalog number: 15524-010 )
  4. 10% ammonium persulfate (Sigma-Aldrich, catalog number: A3678-100G ) (see Recipes)
3. 2x RNA loading dye (see Recipes)
DE81-based nucleic acid purification
1. Fiber glass (Corning Incorporated, catalog number: 988-10144 )
2. Extra thick blot paper (Bio-Rad Laboratories, catalog number: 170-3969 )
3. Whatman DE81 ion exchange cellulose chromatography paper (Thermo Fisher Scientific, catalog number: 05-717-1A )
4. GlycoBlue coprecipitant (Life Technologies, catalog number: AM9516 )
5. 100% ethanol
6. 75% ethanol
7. EB buffer (QIAGEN, catalog number: 19086 )
8. Qubit RNA HS Assay Kit for quantification of purified small RNAs (Life Technologies, catalog number: Q32852 )
9. Qubit dsDNA HS Assay Kit for quantification of DNA amplicon (Life Technologies, catalog number: Q32854 )
10. Low salt buffer (see Recipes)
11. High salt buffer (see Recipes)
Multiplexed small RNA libraries preparation
1. 8-Strip PCR thin-walled, 200 µl tubes (Corning Incorporated, Axygen^®, catalog number: PCR-0208-CP-C )
2. miRNA cloning linker 1 (/5'App/CTGTAGGCACCATCAAT/3'ddC/; 1 nm) (Integrated DNA Technologies, catalog number: 11-04-03-05 )
3. Truncated, K227Q mutation T4 RNA ligase 2 (New England Biolabs, catalog number: M0351L )
4. De-adenylase (New England Biolabs, catalog number: M0331S )
5. Exonuclease VII (United State Biological, catalog number: 70082Z )
6. RNA 5’ adapter (GUUCAGAGUUCUACAGUCCGACGAUC) (Illumina RA5, catalog number: 15013205 )
7. dATP (Life Technologies, catalog number: 55082 )
8. T4 RNA ligase I (Life Technologies, catalog number: AM2141 )
9. RT-PCR primer (ATTGATGGTGCCTACAG; 25 nmol; de-salted) (Integrated DNA Technologies)
10. SuperScript III (Life Technologies, catalog number: 18080051 )
11. Phusion high fidelity II (Thermo Fisher Scientific, catalog number: F549L )
12. 5' PCR primer (Illumina small RNA PCR primer 2: AATGATACGGCGACCACCGACAGGTTCAGA-GTTCTACAGTCCGA)
13. 3' indexed PCR primer I1 - I12 (100 nmol; PAGE-purified) (Integrated DNA Technologies)
  Note: The barcodes below (underlined sequences) are reverse complemented in the final Illumina output sequence (see sequence in square brackets for each primer).
  I1 [CGATGT]: CAAGCAGAAGACGGCATACGAACATCGATTGATGGTGCCTACAG
  I2 [GATCAC]: CAAGCAGAAGACGGCATACGAGTGATCATTGATGGTGCCTACAG
  I3 [CAGATG]: CAAGCAGAAGACGGCATACGACATCTGATTGATGGTGCCTACAG
  I4 [TACGTT]: CAAGCAGAAGACGGCATACGAAACGTAATTGATGGTGCCTACAG
  I5 [TTACCA]: CAAGCAGAAGACGGCATACGATGGTAAATTGATGGTGCCTACAG
  I6 [ACTGTA]: CAAGCAGAAGACGGCATACGATACAGTATTGATGGTGCCTACAG
  I7 [ATCACG]: CAAGCAGAAGACGGCATACGACGTGATATTGATGGTGCCTACAG
  I8 [ACTTGT]: CAAGCAGAAGACGGCATACGAACAAGTATTGATGGTGCCTACAG
  I9 [GCCAAT]: CAAGCAGAAGACGGCATACGAATTGGCATTGATGGTGCCTACAG
  I10 [TGCTAG]: CAAGCAGAAGACGGCATACGACTAGCAATTGATGGTGCCTACAG
  I11 [CTTGTA]: CAAGCAGAAGACGGCATACGATACAAGATTGATGGTGCCTACAG
  I12 [TCAGGC]: CAAGCAGAAGACGGCATACGAGCCTGAATTGATGGTGCCTACAG
14. QIAquick PCR Purification Kit (QIAGEN, catalog number: 28106)
15. Diethylpyrocarbonate (DEPC)-H₂O/RNase-free H₂O
16. DNA size marker suitable for detecting a ~120 bp fragment (50 bp step ladder, Promega Corporation, catalog number: G4521 ; 25 bp step ladder, Promega Corporation, catalog number: G4511 )
17. 3 M sodium acetate (NaOAc) (pH 5.5)
18. Reagents for 6% native PAGE
  1. 37.5:1 polyacrylamide:bisacrylamide (see Recipes)
  2. TEMED (Life Technologies, catalog number: 15524-010)
  3. 10% ammonium persulfate (Sigma-Aldrich, catalog number: A3678-100G) (see Recipes)

Equipment

Thermocycler
Microcentrifuge: For use at room temperature and at 4 °C
Fisher gel-loading tips (Thermo Fisher Scientific, catalog number: 02-707-182 )
Agarose gel electrophoresis systems (Bio-Rad Laboratories, Sub-Cell GT system)
Polyacrylamide gel electrophoresis system (Thermo Fisher Scientific, owl Dual-Gel vertical electrophoresis systems)
65 °C water bath
Semi-dry blotting system (Sigma-Aldrich, semi-dry blotter, catalog number: Z340502 )
Heating block

Procedure

English

中文翻译

文章信息

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如何引用

Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

Gilbert, K. B., Fahlgren, N., Kasschau, K. D., Chapman, E. J., Carrington, J. C. and Carbonell, A. (2014). Preparation of Multiplexed Small RNA Libraries from Plants. Bio-protocol 4(21): e1275. DOI: 10.21769/BioProtoc.1275.
Carbonell, A., Takeda, A., Fahlgren, N., Johnson, S. C., Cuperus, J. T. and Carrington, J. C. (2014). New generation of artificial MicroRNA and synthetic trans-acting small interfering RNA vectors for efficient gene silencing in Arabidopsis. Plant Physiol 165(1): 15-29.