12 A Practical Guide to Cancer Systems Biology
Figure 1. Schematic representation of library construction for transcriptome analysis.
The step-by-step protocol is included in this chapter: (a) Purify and fragment RNA,
(b) RT-PCR: first strand cDNA synthesis, (c) RT-PCR: second strand cDNA synthesis,
(d) repair ends, (e) adenylate 3′ends, (f) ligate adapters, (g) PCR amplification, (h) Gel
purification of amplified library, (i) validate the library.
In the past decade, several parallel NGS platforms have been developed
to provide low-cost, high-throughput sequencing.3,4,9 In this chapter, we
describe the sample preparation in detail for the most commonly used
platform in research and clinical laboratories, the Illumina platform, as
illustrated in Fig. 1.
Consumables and equipment
TruSeq RNA sample Preparation v2 Kit (Illumina, RS-122-2001)
1.5 mL RNase/DNase-free non-sticky tubes (Life Technologies, AM12450)
10 μL, 200μL, and 1000μL barrier pipette tips (General lab supplier)
10 μL, 200μL, and 1000μL single channel pipettes (General lab supplier)
Nuclease-free ultra pure water (General lab supplier)
Ethanol 200 proof (absolute) for molecular biology (Sigma-Aldrich, E7023)
RNase/DNase-free PCR tubes (General lab supplier)
RNase/DNase zapper (General lab supplier)
SuperScript II Reverse Transcriptase (Invitrogen, 18064-014)