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Tissue Collection - Library Preparation

Fresh tissue to cDNA Libraries

Overview
The first step in RNA-Seq sample preparation is to choose a tissue from which you will extract total RNA. Following total RNA extraction you will purify mRNA that will be used as templates to synthesize complementary DNA, also known as cDNA. These resulting cDNA libraries will be used for sequencing.

When selecting the tissue it is important to be consistent in your sampling of different individuals/treatments/locations. Keep in mind that gene expression and SNP detection will be affected by both environmental conditions and tissue type. Before starting the lab work, it is important to note that RNA is highly susceptible to degradation by RNAse enzymes. These enzymes can be found in the air and on your clothing and hands. It is very important to utilize good laboratory practices and work as efficiently as possible through cDNA synthesis. Once you have double stranded cDNA you can breathe a little easier, because cDNA is more stable than RNA. While there are many RNA extraction kits to choose from we recommend using Qiagen's RNeasy extraction kit. It is a very fast and easy procedure, free from toxic chemicals. However, if you plan to also extract genomic DNA from your tissue, a Trizol extraction method may be preferable. While there are also many cDNA library preparation kits, Illumina's TruSeq RNA sample preparation kit is all-inclusive. It includes all the necessary reagents to purify mRNA from total RNA and make cDNA libraries. The protocol is straightforward and there is peace of mind knowing the manual was written and tested by the manufacturers of the Illumina sequencing machine.

Objectives
The objectives for this section are to 1) extract total RNA from fresh, frozen, or RNAlater- preserved tissue samples, 2) purify and fragment mRNA from total RNA and, 3) create cDNA libraries from mRNA that will be sent off for sequencing.

Resources
Gayral P, Weinert L, Chiari Y, Tsagkogeorga G, Ballenghien M, Galtier N. 2011. Next-generation sequencing of transcriptomes: a guide to RNA isolation in nonmodel animals. Molecular Ecology Resources 11: 650-661.

Collins L, Biggs P, Voelckel, C, Joly, S. 2008. An approach to transcriptome analysis of non-model organisms using short-read sequences. Genome Informatics 21: 3-14.

Mortazavi AWilliams BAMcCue KSchaeffer LWold B. 2008. Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nature Methods. 5: 621-628.

RNA extraction kits and protocols
http://www.qiagen.com/Products/Catalog/Sample-Technologies/RNA-Sample-Technologies/Total-RNA/RNeasy-Mini-Kit#resources

Quantification/validation of samples
http://www.invitrogen.com/site/us/en/home/brands/Product-Brand/Qubit.html

Library Prep/RNA seq technology
http://www.illumina.com/documents//products/datasheets/datasheet_TruSeq_sample_prep_kits.pdf
http://www.illumina.com/TruSeq.ilmn

Process
1. Use Qiagen's RNeasy Kit (http://www.qiagen.com/Products/Catalog/Sample-Technologies/RNA-Sample-Technologies/Total-RNA/RNeasy-Mini-Kit#resources) or any other RNA extraction protocol to extract total RNA from your tissue.

  • A tissue-lyser is a great way to disrupt the tissue sample.

2. Quantify the amount of total RNA in each sample using a QuBit RNA assay. (http://products.invitrogen.com/ivgn/product/Q32852).

  • You will want to standardize across samples the amount of total RNA you use to start

            library preparation. 1 µg of total RNA is often a good amount to start with.

    1. Flash-freeze the freshly extracted total RNA in liquid nitrogen and place at -80°C until ready to proceed with library preparation.
    2. Prepare libraries following the Illumina TruSeq Preparation Kit protocol (www.illumina.com/.../datasheet_TruSeq_sample_prep_kits.pdf)

Summary

You have now extracted total RNA from a tissue, purified mRNA from the total RNA, and from this mRNA have created cDNA libraries that are ready for sequencing. Congratulations! You are well on your way to collecting a single data set that contains both gene expression and SNP data from the tissue you have selected.

Tissue   Sequencing   Computer   QC   Assembly   Annotation   Mapping   Expression   SNP

De Wit P, Pespeni MH, Ladner JTBarshis DJ, Seneca F, Jaris H, Overgaard Therkildsen N, Morikawa M and Palumbi SR (2012) The simple fool's guide to population genomics via RNA-Seq: an introduction to high-throughput sequencing data analysis.  Molecular Ecology Resources 12, 1058-1067.