Protein G magnetic Dynabeads (Invitrogen) were prepared with IPP buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.1% NP-40). 2.5 μg of anti-m7G antibody (SYSY, catalog nr: 201 011) were incubated with the beads for at least 1 hour in 4ºC. The beads were finally washed twice with IPP buffer. RNA extractions were carried out with the Direct-zol Miniprep kit (Zymo Research). Abundant small RNAs (< 200 nt) were removed using the RNA Clean and Concentrator Kit (Zymo Research) and eluted in 100 μl TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA). RNA fragmentation was performed using the Covaris E220 Focused-ultrasonicator (Duty cycle: 10%, Intensity: 5, Power: 175 W, Cycles/Burst: 200, time: 140 s). After sonication, the capped RNA was captured with the antibody-coupled Protein G magnetic beads for 1 to 2 hours in 4 ºC. Then, the beads were washed 3 times with IPP buffer. RNA 3' ends were repaired using PNK. Custom-made, barcoded adapters were ligated to the RNA using T4 RNA Ligase 1 for 1 h at 25 °C. Excess adapters were washed away with IPP buffer and RNA was purified by column purification. Isolated RNA was reverse-transcribed and treated with RNase H. cDNA was column-purified and circularized with CircLigase for 2–16 hours. Circularized cDNA was directly PCR amplified, the amplified library was finally cleaned up using Ampure beads.
GEO Accession
GSM3344579