- Low molecular DNA weight marker (e.g., NEB).
- SYBR safe.
- Circligase II, MnCl 2 , and 10buffer.
- Fast Digest BamH1.
- PCR mastermix (e.g., Thermo Fisher Accuprime supermix I).
- Illumina qPCR library quantification kit (e.g., Kapa
Biosystems). - 4–12% protein denaturing precast gels (e.g., Thermo Fisher
NuPAGE) (seeNote 2). - 6% TBE-UREA precast gels (e.g., Thermo Fisher).
- 6% TBE precast gels (e.g., Thermo Fisher).
2.3 Required
Oligonucleotides
- L3-IR-app adapter: /5Phos/AGATCGGAAGAGCGGTTCA
GAAAAAAAAAAAA/iAzideN/AAAAAAAAAAAA/3Bio/
Adapter requires adenylation then click chemistry conjugation
of the IRDye 800CW DBCO infrared dye (Li-Cor) before use. - RT-primers: /5Phos/NNAACCNNNAGATCGGAAGAGCG
TCGTGgatcCTGAACCGC
underlined nucleotides can be replaced with different index
barcodes e.g., RT1: AACC, RT2: ACAA, RT3: ATTG, RT4:
AGGT, RT6: CCGG, RT7: CTAA, RT8: CATT. - Cut4oligo: 5^0 GTTCAGGATCCACGACGCTCT TCaaaa.
- P5 primer: 5^0 AATGATACGGCGACCACCGAGATCTACA
CTCTTTCCCTACACGACGCTCTTCCGATCT. - P3 primer: 5^0 CAAGCAGAAGACGGCATACGAGATCGGT
CTCGGCATTCCTGCTGAACCGCTCTTCCGATCT.
2.4 Buffers 1. Lysis Buffer: 50 mM Tris–HCl, pH 7.4, 100 mM NaCl, 1%
Igepal CA-630, 0.1% SDS, and 0.5% sodium deoxycholate.
Sterile filter and store at 4C.
- High-Salt Wash: 50 mM Tris–HCl, pH 7.4, 1 M NaCl, 1 mM
EDTA, 1% Igepal CA-630, 0.1% SDS, and 0.5% sodium deox-
ycholate. Sterile filter and store at 4C. - PNK Buffer: 20 mM Tris–HCl, pH 7.4, 10 mM MgCl 2 , 0.2%
Tween-20. Sterile filter and store at 4C. - 5PNK pH 6.5 Buffer: 350 mM Tris–HCl, pH 6.5, 50 mM
MgCl 2 , and 5 mM dithiothreitol. Use nuclease-free H 2 O.
Dispense to 50μL aliquots and store at 20 C. Only use
aliquots once. - 4 Ligation Buffer: 200 mM Tris–HCl, pH 7.8, 40 mM
MgCl 2 , and 4 mM dithiothreitol. Use nuclease-free H 2 O.
Dispense to 50μL aliquots and store at 20 C. Only use
aliquots once.
iCLIP to Determine Protein-RNA Interactions 435