Author: Ian M. Silverman; Sager J. Gosai; Nicholas Vrettos; Shawn W. Foley; Nathan D. Berkowitz; Zissimos Mourelatos; Brian D. Gregory
Title: Isolation and sequencing of AGO-bound RNAs reveals characteristics of mammalian stem-loop processing in vivo Document date: 2018_4_6
ID: 1pbshnw9_30
Snippet: RNA was purified from AGO immunoprecipitates, dephosphorylated and labeled with P 32 -γ-ATP. Autoradiography of the RNA gel showed a major band at 20-25 nt, representing mature miRNAs, and several other prominent bands between 50-80 nt, corresponding to the size range of pre-miRNAs (Fig. 1B) . We excised gel slices from both of these regions and generated high-throughput sequencing libraries, referred to herein as miRNA-seq and pre-miRNA-seq lib.....
Document: RNA was purified from AGO immunoprecipitates, dephosphorylated and labeled with P 32 -γ-ATP. Autoradiography of the RNA gel showed a major band at 20-25 nt, representing mature miRNAs, and several other prominent bands between 50-80 nt, corresponding to the size range of pre-miRNAs (Fig. 1B) . We excised gel slices from both of these regions and generated high-throughput sequencing libraries, referred to herein as miRNA-seq and pre-miRNA-seq libraries, respectively. Previous attempts to sequence pre-miRNAs have been unsuccessful due to the inaccessibility of the pre-miRNA 5' ends. Therefore, we used a method that attaches the 5' linker through a CircLigase-mediated cDNA circularization step (see Methods) (19, 29) .
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