- Transcriptome Analysis: Library Construction 13
Agencourt AMPure XP 60 mL kit (Bechman Coulter Genomics, A63881)
Tris-HCl 10 mM, pH8.5 with 0.1% Tween 20 (General lab supplier)
Tween 20 (Sigma-Aldrich, P7949)
Agilent 2100 Bioanalyzer (Agilent Technologies Inc., G2939AA)
Magnetic stand (Life Technologies, 12321D)
Thermal cycler with heated lid (General lab supplier)
Centrifuge (General lab supplier)
Vortexer (General lab supplier)
Preparation
Thaw the following items to room temperature (25◦C) for 30 min before use.
— RNA Purification Beads (RPB) — End Repair Control (CTE)
— Bead Binding Buffer (BBB) — End Repair Mix (ERP)
— Bead Washing Buffer (BWB) — A-Tailing Control (CTA)
— Elution Buffer (ELB) — A-Tailing Mix (ATL)
— Elute, Prime, Fragment Mix (EPF) — Ligation Control (CTL)
— First Strand Master Mix (FSM) — RNA Adapters Indices
— Resuspension Buffer (RSB) — Stop Ligation Buffer (STL)
— Second Strand Master Mix (SSM) — PCR Master Mix
(PMM, on ice)
— AMPure XP Beads — PCR Primer Cocktail
(PPC, on ice)
I.Check RNA quality
Before proceeding with library construction, sample RNA quality and
integrity should be checked by an Agilent 2100 Bioanalyzer. An RNA
integrity number (RIN) of at least 8.0 is the recommended threshold.
Figure 2 shows examples of high quality total RNA extracted from human
lung adenocarcinoma cell A549 using conventional Trizol protocol and
treated with DNase I. Use of 0.1–4μg of total RNA for transcriptome library
construction is recommended.
II.Purify and fragment mRNA
- Dilute the total RNA with nuclease-free ultra pure water (NFW) to a final
volume of 50μLinaPCRtube.