- Nonlabeled/“light” cell culture medium: mix 0.5 L of
Dulbecco’s modified Eagle’s medium (DMEM) containing
high glucose (4.5 g/L), 50 mL of fetal bovine serum, 5 mL
of 200 mM L-glutamine, and 5 mL of 10,000 U/mL
penicillin-streptomycin. Store at 4C. - 0.5 M 4-thiouridine (4SU) solution: Dissolve 1 g of 4SU
(ChemGenes) in 7.68 mL of deionized water. Aliquot and
store at 20 C protected from light. - Phosphate buffered saline (PBS). Store at 4C.
- 5 M lithium chloride solution. Dissolve 106 g of LiCl powder
by heating and agitation in 0.5 L of deionized water and store
at 4C. - 20 w/v % lithium dodecyl sulfate (LiDS) solution. Wear pro-
tective mask and glasses when handling LiDS powder. Dissolve
100 g of LiDS powder with agitation in 0.5 L of water and
store at room temperature. - IGEPAL CA-630 (NP-40 substitute). Store at room
temperature. - Lysis/binding buffer: 100 mM Tris, pH 7.5, 500 mM LiCl,
10 mM EDTA, 1 w/v % lithium-dodecylsulfate/LiDS, 5 mM
DTT, and 1complete Mini EDTA-free protease inhibitor
cocktail. Store at room temperature. Immediately before use
add 0.25 mL of 1 M DTT and one tablet of complete mini
EDTA-free protease inhibitor cocktail (Roche) per 50 mL of
working lysis/binding buffer. - Washing buffer: 50 mM Tris, pH 7.5, 140 mM LiCl, 2 mM
EDTA, 0.5% IGEPAL-CA630, 0.5 mM DTT, and 1com-
plete mini EDTA-free protease inhibitor cocktail. Store at 4C.
Immediately before use add 25μL of 1 M DTT and one tablet
of complete Mini EDTA-free protease inhibitor cocktail
(Roche) per 50 mL of working buffer. - Magnetic oligo(dT) beads (e.g., Thermo Fisher Scientific
Oligo(dT) 25 Dynabeads, component of the mRNA DIRECT
kit). - Low-salt elution buffer: 10 mM Tris, pH 7.5. Store at 4C.
The elution buffer provided in the mRNA DIRECT kit
(Thermo Fisher Scientific) may also be used. - Concentrating buffer: 10 mM Tris, pH 7.5, 50 mM NaCl.
Store at 4C. - 100 U/μL RNase I solution. Store at 20 C.
- 125 U/μL benzonase solution. Store at 20 C.
- 1 M MgCl 2. Dissolve 4.76 g of MgCl 2 in 50 mL of deionized
water. Store at 4C.
408 Miha Milek and Markus Landthaler