Alternative polyadenylation of MCF7
1) RNA fragmentation. About 200 ng total RNA for each sample was randomly fragmented with heating at 80℃ for 30 minutes. 2) The first round of reverse transcription. An anchored oligo d(T) tagged with Illumina A adaptor and T7 promoter was used for the first strand cDNA synthesis, and then the second strand was synthesized with RNase H, E. coli DNA polymerase and DNA ligase. The frayed termini of double-stranded cDNA were polished with T4 DNA polymerase. Subsequently, the products were purified with Agencourt RNAClean XP kit (Beckman Coulter). 3) In vitro transcription. RiboMAX™ Large Scale RNA Production System-T7 (Promega) was used to perform in vitro transcription with the manufacturer's manual, and then the template cDNA was removed with RQ1 RNase-Free DNase (Promega). The RNA product was also purified with Agencourt RNAClean XP kit (Beckman Coulter). 4) The second round of reverse transcription. Random primers tagged with part of Illumina B adaptor were used for the first strand cDNA synthesis, and then the product was purified with MinElute PCR Purification Kit (Qiagen). 5) PCR amplification. Illumina adaptor B with 6-mer barcode and modified oligo d(T) tagged with Illumina adaptor A were used to perform the PCR amplification. Finally, size selection of 200-500bp fragments of the PCR product was performed with AMPure XP Beads (Beckman Coulter), and then the quality of the library was checked with Agilent 2100 Bioanalyzer.
Sun Yat-sen University
MCF7