Oligotex mRNA Midi Kit (12)

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	Follow manufacturer’s instructions

Protocol tips
Follow manufacturer’s instructions

Publication protocol

Libraries 1–3 were generated from 4–6-week-old inflorescence tissue, and library 4 was generated from 10-day-old Col-0 A. thaliana seedlings. Total RNA was isolated with TRI Reagent (Sigma) per the recommended protocol followed by polyA+ RNA purification with the Oligotex mRNA Midi Kit (QIAGEN) via the batch protocol. Five micrograms of polyA+ RNA was added to a 50 DNA-RNA hybrid adaptor containing a 30 MmeI site at a ratio of 136 pmol/mg of polyA+ RNA to set up the 50 adaptor ligation. The polyA+ RNA, water, and adaptor were combined and heated at 65􏰀C for 5 min and then transferred to ice for 2 min. T4 RNA ligase buffer and T4 RNA ligase (New England Biolabs) were added for a 0.5 or 1 hr incubation at 37􏰀C. Li- brary 2 underwent a second polyA+ purification after the 50 adaptor ligation, and libraries 3–4 were passed through MicroSpin S-300 columns (GE Healthcare) to remove unligated adaptor. Library 2 was reverse transcribed with the GeneRacer oligo(dT) primer (Invitrogen GeneRacer) and pool ampli- fied. First-strand cDNA was generated in libraries 3–4 with random hexam- ers added at a ratio of 83.3 ng/mg mRNA, following the RT-PCR protocol from [S1]. After reverse transcription, libraries 3–4 were eluted in 50 mM NaOH, 5 mM EDTA (pH 8) to degrade remaining RNA. After 1 hr at 65􏰀C, an equal volume of 1M Tris-HCl (pH 7.5) was added to neutralize the reac- tion. Then, libraries 3–4 were passed through G-25 microspin columns (GE Heathcare) and primer extended. All libraries underwent the MmeI di- gest with the same conditions with 2U of MmeI being added per mg of library followed by a 1 hr incubation at 37􏰀 C. Two oligos were annealed to make a 30 dsDNA adaptor. One hundred picomoles of this adaptor was added per mg of library along with 400U of T4 DNA Ligase (New England Biolabs) in a 5 hr room-temperature incubation. The library was purified with an 8% (19:1 acryl:bis-acryl) gel and then amplified. The amplified library then underwent a final gel purification prior to sequencing.

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Manufacturer protocol

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