Isolation and sequencing of AGO-bound RNAs reveals characteristics of mammalian stem-loop processing in vivo

Ian M. Silverman; Sager J. Gosai; Nicholas Vrettos; Shawn W. Foley; Nathan D. Berkowitz; Zissimos Mourelatos; Brian D. Gregory

RNA was dephosphorylated with antarctic phosphatase (NEB) and P 32 -γ-ATP labeled with T4 polynucleotide kinase (NEB). 5' end radiolabeled RNAs were resolved on a 7 M urea, 15% polyacrylamide gel. Gel slices from 20-25 nt (miRNA-seq) and 50-80 nt (pre-miRNA-seq) were recovered and ligated to miRCat 3' Linker-1 (IDT) with T4 RNA Ligase 2 Truncated (NEB), in the presence of 15% PEG8000. Ligation products were PAGE purified and reverse transcribed by a 5' phosphorylated RT primer containing 3' and 5' adaptor complementary sequences. To do this, RT primer . CC-BY 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/294488 doi: bioRxiv preprint was mixed with RNA and heated at 80°C for 5 min, then incubated at 60°C for 5 min and finally cooled down to room temperature before the remaining reagents were added (Affinityscipt, Stratagene) (31) . cDNAs were resolved and purified from a 7 M urea, 6% polyacrylamide gel, then circularized with CircLigase I (Epicentre) and PCR amplified with Phusion DNA polymerase (NEB). The amplicon yield was boosted with a second PCR amplification round, then size selected on a 3% metaphor gel (Lonza), purified, and sent for deep sequencing at the Next-Generation Sequencing Core at the University of Pennsylvania. All oligonucleotide sequences used in this study can be found in the attached document (Table S9) .