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cDNA library construction

AS Aijun Sun
XZ Xiaojing Zhu
YL Ying Liu
RW Rui Wang
SY Shuaikang Yang
MT Man Teng
LZ Luping Zheng
JL Jun Luo
GZ Gaiping Zhang
GZ Guoqing Zhuang
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RNA samples were fragmented into 100 bp using fragmentation buffer and then incubated with anti-m6A polyclonal antibody (Synaptic Systems, 202,003, Germany) in immunoprecipitation (IP) buffer for 2 h at 4 °C. The mixture was then immunoprecipitated by incubation with protein-A beads (Thermo Fisher Scientific, Waltham, MA, USA) at 4 °C for an additional 2 h. Then, bound RNA was eluted from the beads with N6-monophosphate (BERRY & ASSOCIATES, PR3732) in IP buffer and then extracted with Trizol reagent. Purified RNA was used for RNA-seq library generation with NEBNext® Ultra™ II Directional RNA Library Prep Kit (New England Biolabs, USA) following the manufacturer’s instructions. Both the input sample without immunoprecipitation and the m6A IP samples were subjected to 150 bp paired-end sequencing on an Illumina HiSeq 4000 sequencer [14].

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