- PK Buffer: 100 mM Tris–HCl, pH 7.4, 50 mM NaCl, and
10 mM EDTA. Sterile filter and store at 4C. - PK Buffer +7 M Urea: 100 mM Tris–HCl, pH 7.4, 50 mM
NaCl, 10 mM EDTA, and 7 M Urea. Sterile filter and store at
4 C. - 1NuPAGE MOPS-SDS buffer: Add 25 mL of 20MOPS-
SDS buffer to 475 mL of water. - 1NuPAGE loading buffer (per sample): 5μL4NuPAGE
Loading Buffer, 2μL sample reducing reagent, and 13μL
nuclease-free H 2 O.
2.5 Reaction Mixes 1. PNK de-phosphorylation mix (per sample): 15μL nuclease-
free H 2 O, 4μLof5PNK pH 6.5 Buffer, 0.5μL PNK, and
0.5μL RNAsin.
- Ligation mix (add reagents in the indicated order): 8.5μL
nuclease-free H 2 O, 5μLof4ligation buffer, 0.5μLT4
RNA ligase, 0.5μL RNAsin, 1.5μL1μM L3-IR-app oligo,
and 4μL PEG 400. - Reverse transcription mix (per sample): 7 μL nuclease-free
H 2 O, 4μL5RT buffer, 1μL 0.1 M DTT, 0.5μL RNasin,
0.5μL Superscript III. - Circularization mix (per sample): 6.5μL nuclease-free H 2 O,
0.8μL10CircLigase II buffer, 0.4μL 50 mM MnCl 2 , and
0.3μL CircLigase II. - Cut oligo mix (per sample): 25μL nuclease-free H 2 O, 4μL
FastDigest buffer, and 1μL10μM cut oligo. - Test-PCR mix (per sample): 3.75μL ddH 2 O, 5μL AccuPrime
Supermix 1, 0.25μL10μM P5/P3 Solexa primer mix. - Final-PCR mix (per sample): 9μL ddH 2 O, 20μL AccuPrime
Supermix 1, and 1μL10μM P5/P3 Solexa primer mix. - Universal qPCR Master mix (per sample): 12.4 μL qPCR
Master mix (Primer Premix and ROX High/Low should be
added to Master Mix prior to first use as per manufacturer’s
instructions), 3.6μL ddH 2 O.
3 Methods
3.1 Sample
Collection
- Remove media from cells growing at 80–90% confluency on a
10 cm dish and replace with 6 mL of ice-cold PBS (seeNote 3).
Important: Samples should remain at 4 ̊C from this point
forward unless indicated.
Optional: To test if the signal is dependent on expected
antigen and not a contaminant RBP, use a control cell-line
in which the RBP of interested is knocked down or
knocked out (seeNote 4).
436 Christopher R. Sibley