GSM2416963: HEK293T-Wt NAD-Capture 1; Homo sapiens; RNA-Seq
Total RNA was isolated by TRIzol extraction, following the manufacturers recommended protocol enrichment protocol: RiboMinus enrichment for non-ribosomal RNAs was performed as recommended by the kit manufacturer enrichment protocol: NAD-Capture enrichment was performed using the NAD-RNA Capture protocol (Cahova et al., 2015) with minor modification.  NAD-Capture was carried out with 100µg total RNAs treated with 10µl 4-pentyn-1-ol (Sigma-Aldrich) and 1.5U Adenosine diphosphate-ribosylcyclase (ADPRC) in 100µl reaction containing 50mM HEPES, 5mM MgCl2 (pH 7) and 40U RNasin® Ribonuclease Inhibitor (Promega) at 37oC 60 minutes. The reaction minus ADPRC was used as background control. The reaction was stopped with phenol/chloroform extraction and RNAs were precipitated with ethanol. The precipitated RNAs were subjected to copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction by incubating with 250µM biotin-PEG3-azide, freshly mixed 1mM CuSO4, 0.5mM THPTA, 2mM Sodium Ascorbate in 100µl reaction with 50mM HEPES, 5mM MgCl2 (pH7) and 40U RNasin® Ribonuclease Inhibitor (Promega) at 30oC for 30 minutes. Following extraction with phenol/chloroform and RNA precipitation, the NAD clicked RNAs were captures by binding to 20µl streptavidin magnet beads at room temperature with gentle rocking for 1 hour in 100µl binding buffer (1M NaCl, 10mM HEPES (pH7) and 5mM EDTA). The beads were washed three time with wash buffer (8mM Urea, 50mM Tris-HCl (pH7.4), 0.25% Triton-X100) and three washes with nuclease free H2O. RiboMinus-selected RNAs were reverse transcribed and converted to libraries using the Illumina TruSeq RNA Sample Preparation Kit v2. For NAD-Capture RNAs, samples were reverse transcribed into cDNA and synthesized into dsDNA with the SMARTer® Universal Low Input RNA Kit for Sequencing (Clontech Laboratories, Inc.) following the manufacturer’s protocol.
GEO Accession
GSM2416963