RNA Circularization for 5’ and 3’ end mapping
Begin with 1ug total RNA
Decapping
1. Assemble reaction:
 1ug RNA
 20 U Tobacco Acid Pyrophosphatase (TAP)
 10X TAP buffer
 H2O to 10ul
2. Incubate at 37° for 1hr.
3. Purify over ProbeQuant G-50 Micro Column
Ligation
1.Assemble reaction
 RNA (50ul from column purification)
 RNaseOUT (2ul)
 100U T4 RNA ligase (20ul)
 10X buffer (10ul)
 DMSO (10ul)
 H2O to 100ul (8ul)
2. Incubate at 4°, overnight (16 hr)
3. Purify over ProbeQuant G-50 Micro Column
cDNA Synthesis (Invitrogen superscript system)
1. Thaw 5X first strand buffer immediately before reaction and re-freeze immediately.
2. Add directly to ligated RNA:
 1 μl specific primer*
 2 μl 10 mM dNTP
 4ul H2O
3. Heat to 65o for 5 min., chill on ice.
4. Spin down quickly and add:
 16 μl 5X first-strand buffer
 5 μl 0.1 M DTT
 2 μl RNAseOUT
5.
6.
7.
8.
9.
Mix gently and incubate at 42o C for 2 min. (Total volume 80ul)
Add 2 μl (200 units) of Superscript II RT, mix by pipetting gently.
Incubate at room temp for 10 min.
Incubate at 42o C for 50 min.
Inactivate reaction by heating to 70o for 15 min.
*Specific primer for cDNA should be designed approximately 700 bp from 5’ end in
reverse orientation (running towards 5’ end of transcript of interest)
Analysis of Circularized Products
Design primers that flank the junction region. These should run towards the 5’ and 3’
ends of the transcript of interest. It’s a good idea to design nested primer sets if your
transcript is not abundant. Simply run a PCR on your circularized cDNA, subclone
fragments of the expected size, and sequence.