Library |
Library name |
Construction protocol |
Strategy |
Source |
Selection |
Layout |
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mRNAs were purified using Dynabeads mRNA Purification Kit (Life Technologies, 61006) and fragmented to size around of 100 nt using the fragmentation reagent (Life Technologies, AM8740). 10 μg of purified mRNAs were mixed with 25 μg of anti-m6A antibody (Abcam, ab151230) in 450 μl immunoprecipitation buffer (50 mM Tris, pH 7.4, 100 mM NaCl, 0.05% NP-40) and incubated by rotating at 4 °C for 2 h. The solution was then transferred to a clear flat-bottom 96-well plate (Corning) on ice and irradiated three times with 0.15 J/cm2 at 254 nm in a CL-1000 Ultraviolet Crosslinker (UVP). The mixture was then immunoprecipitated by incubation with Dynabeads Protein A (Life Technologies, 1001D) at 4 °C for 2 h. After extensive washing and on-bead end-repair and linker ligation, the bound RNA fragments were eluted from the beads by proteinase K digestion at 55 °C for 1 h. RNAs were isolated by further phenol-chloroform extraction and ethanol precipitation. Purified RNAs were used to construct the library using SMARTer smRNA-Seq Kit for Illumina (Takara, 635029) according to the manufacturer’s instructions. Sequencing was carried out on Illumina HiSeq X-ten platform with paired-end 150 bp read length. |
RNA-Seq |
TRANSCRIPTOMIC |
PCR |
PAIRED
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