no treatment control for POST5
RNA was randomly fragmentated by ZnCl2. After separation on TBE-Urea PAGE gel, RNA fragments that migrate between 40-70 bp DNA ladder were selected and their 3 ends were dephosphorylated by T4 PNK. The miRNA cloning liker 2 (5App/CACTCGGGCACCAAGGA/3ddC) was used as the universal 3 linker and pre-adenylated using the 5 DNA Adenylation Kit with provided protocol. The resutling RNA was reverse transcriped using TGIRT reverse transcriptase and subsequently self-circularized by Circ ligase. The self-ligated ssDNA was amplified by Phusion DNA polymerase.