- RNeasy Plus Mini Kit (Qiagen,seeNote 1).
- mRNA Miniprep Kit (e.g., Sigma-Aldrich GenElute).
- Gel Extraction Kit (e.g., Qiagen MinElute)
- 4–20%TBE Gels, 10 well.
- 0.5TBE buffer.
- RNA Loading Dye, (2).
- Low Range ssRNA Ladder.
- SYBR Gold Nucleic Acid Gel Stain (10,000Concentrate in
DMSO). - TruSeq Stranded mRNA Library Preparation Kit Set A
(Illumina).
11.β-mercaptoethanol.
- 100% ethanol.
- 70% ethanol, freshly prepared in DEPC-treated nuclease-free
water. - 3 M sodium acetate pH 5.2.
- 2.5% low melting point agarose gel with 0.5μg/mL ethidium
bromide. - 1TAE buffer.
- 50 mg/mL UltraPure BSA.
- PBS, sterile.
- Protein A beads for Immunoprecipitation (e.g., Thermo
Scientific Dynabeads). - SUPERase In RNase Inhibitor (20 U/μL) (Ambion).
- Agencourt AMPure XP Beads (Beckman Coulter).
- FastStart Essential DNA Green Master (Roche).
- 5IP buffer: 50 mM Tris–HCl, pH 7.4, 750 mM NaCl, and
0.5% NP-40. - Wash buffer (kept on ice, prepared fresh): 5IP buffer diluted
to 1in nuclease-free water, supplemented with 0.1% SUPER-
ase inhibitor. - 20 mM m^6 A solution: dissolve 10 mg of m^6 A monophosphate
sodium salt in 1.3 mL of DEPC-treated nuclease-free water.
Aliquot and store at 20 C; use within 12 months. - m^6 A-specific antibody solution, 0.5 mg/mL: reconstitute
50 μg of affinity purified anti-m^6 A rabbit polyclonal antibody
(Synaptic Systems, cat. no. 202003) in 100μL of DEPC-
treated nuclease-free water. Aliquot and store at 20 C;
avoid multiple freeze–thaw cycles; use within 12 months. - Blocking buffer: wash buffer supplemented with 0.5 mg/mL
UltraPure BSA.
Identifying the m^6 A Methylome 51