GSM1843477: HEK293T pre-miRNA-seq; Homo sapiens; miRNA-Seq
Pre-miRNA-seq and miRNA-seq was performed as previously described [18]. Briefly, HEK293T cells were lysed in RSB 200 (20 mM Tris-HCl pH 7.4, 200 mM NaCl, 2.5 mM MgCl2, 0.5% NP-40) supplemented with 0.2 U/μl RNaseIn  (Promega) and 1tab/10ml of protease inhibitor cocktail (Roche) with 1-3 10s bursts of sonication. Cleared lysates were incubated with agarose protein G beads (Life Technologies) conjugated to 12μg of AGO 2A8 antibody or 3 μl of non-immune serum (NIMS) per 1 ml of lysate. Conguated beads were incubated with lysate for 1.5 h at 4°C on nutator and washed 4X with lysis buffer. 500 μl of trizol was added to washed beads and vortexed for 30 s. 150 μl of chloroform was added and the reaction and vortexed for 30 s followed by 20 m of centrifugation at 16,000 g.  The supernatant was transferred to a new tube with 300 μl of isopropanol and 15 μg of glycogen. Pellets were recovered and RNA was dephosphorylated with phosphatase and labeled with T4 polynucleotide kindase and λ-p32-ATP. 5’ end radiolabeled RNA from AGO-IPs were resolved on a 15% denaturing PAGE gel with 7M urea. Gel slices from 20-25 nt (miRNA-seq) and 50-80 nt (pre-miRNA-seq) were recovered and ligated to miRCat 3’ Linker (IDT) with T4 RNA Ligase 2 Truncated (NEB). Ligation products were resolved on a 15% PAGE gel with 7 M urea, size selected and repurified.  Reverse transcription was performed and product was purified on a 10% PAGE gel and repurified.  cDNA was circularized using CircLigase I and PCR amplified.  PCR amplicons were gel purified on a 3% Metaphor Gel, size selected and a second round of PCR was peformed.  Product was again size selected, repurified and submitted for sequenceing.