GSM4221105: 24-3 24h; synthetic construct; OTHER
Hammerhead ribozyme RNA was synthesized from a corresponding template RNA, a biotinylated RNA primer and the 24-3 or 38-6 RNA polymerase ribozymes at concentrations of 100 nM, 80 nM, and 100 nM, respectively, in a reaction mixture containing 4 mM each NTP, 200 mM MgCl2, 25 mM Tris-HCl (pH 8.3), and 0.05% Tween-20, which was incubated at 17 °C for 24 h for 24-3 and 1 h for 38-6. Extended RNA primers were captured on MyOne Streptavidin C1 dynabeads, washed to remove RNA template and polymerase ribozyme, and recovered by heating in 95% formamide and 10 mM EDTA at 95 °C for 10 min, then ethanol precipitated. The purified RNAs were ligated to the NEB Universal miRNA cloning linker using K227Q T4 RNA ligase 2 according to the manufacturer protocols, reverse transcribed using Superscript IV (ThermoFisher), and the cDNA poly(C)-tailed using Terminal Transferase and a standard protocol (New England Biolabs). All enzymes were heat inactivated at 80 °C for 20 min, prior to library construction from cDNA by nested PCR using the Q5 Hot-Start High Fidelity DNA Polymerase (New England Biolabs), first using primers complementary to the constant flanking regions of the cDNA to append Illumina adapter sequences, then using Illumina Nextera primers to add index sequences (503 for both, 701 and 702 for 24-3 and 38-6 samples, respectively). 150-250nt PCR products were isolated by agarose gel and purified by the PureLink Gel Purification Kit (ThermoFisher) prior to sequencing. Library was composed of two constant regions flanking the variable length and sequence products from the RNA polymerization reactions: 5'-CTACAGGGCACTCCACAC - X - CTGTAGGCACCATCAAT-3', where X is variable, but would be 5' - GACGTACTGATGAGGCCGAAAGGCCGAAAAGCGTTTTTTGTCATTGTC - 3' if the RNA template was copied accurately and completely. Adapter and index sequences were appended to the 5'- and 3'- ends as described in the library construction protocol.
GEO
GSM4221105
GEO Accession
GSM4221105