Genome-Wide Analysis of A-to-I RNA Editing via Single Molecule Sequencing in Drosophila
Total RNA from whole male Canton-S (WT) flies was isolated using TRIzol(R) reagent (Invitrogen) following the manufacturer's protocol.    Total RNA was then treated with DNase I as follows: 20ug of total RNA was mixed with 10ul TurboDNase Buffer (Applied Biosystems); 1ul RnaseOut (Invitrogen); and 2ul TurboDNase (Applied Biosystems) and incubated for 30 minutes at 37C.    The RNA was then purified using the RNeasy MinElute kit (Qiagen).    Samples underwent depletion of rRNA through the use of the RiboMinus Eukaryote Kit for RNA-Seq (Invitrogen, A10837-08) following the manufacturer's protocol modified in the following ways:  Oligos complimentary to the drosophila 18S and 28S rRNA transcript were purchased such that they each had a 5'-biotinylation (IDT) (see below for the list of oligos).    The oligos were resuspended at 1000uM and an equimolar mastermix made.    A total of 2000pmol oligo was added to 19ul DEPC water and the mixture incubated at 95C for 5 minutes then rapidly cooled on ice.    Eight microliters of 10mM ddNTP mixture (Roche), 4ul 2.5mM CoCl, 10x TdT buffer and 3ul Terminal Transferase (NEB) were added to the oligos and incubated at 37C for 1 hour and 70C for 10 minutes.    The oligos were then cleaned twice on Performa DTR cartridges (EdgeBio) following manufacturer's protocols.    The RiboMinus protocol was followed, except that 2.5ul of the prepared oligo mixture was added to the RNA prior to hybridization, when the RiboMinus probe was added.    Up to 1600ng of rRNA-depleted RNA (in eight 200ng aliquots) underwent cDNA synthesis as described above, with the exception that the 85C incubation was replaced with a 10 minute incubation at 70C followed by rapid cooling on ice.    Second strand synthesis was performed by pooling the cDNA synthesis products and adding 1714ul DEPC water, 571ul 5x second strand buffer (Invitrogen), 57ul 10mM dNTPs (Invitrogen), 19.2ul 10U/ul E.coli DNA ligase (Invitrogen), 76ul 10U/ul E.coli DNA polymerase I (Invitrogen) and 19.2ul 2U/ul E.coli RNAseH (Invitrogen).    The resultant mixture was distributed to pcr tubes and incubated at 16C for 2 hours and 70C for 15 minutes.    The ds-cDNA was then purified on QIAQuick PCR cleanup kits (Qiagen) and eluted twice in 20ul TE.    A target concentration of 100-150ng/ul is desired.    Normalization occurs through the denaturation and re-hybridization of the ds cDNA followed by the selective degradation of ds-cDNA by Duplex-specific nuclease (DSN) (Evrogen).    Three microliters of 100-150ng/ul ds cDNA were added to 1ul 4x hybridization buffer (200mM HEPES, pH 7.5, 2M NaCl), mixed thoroughly in a PCR tube and overlaid with 10ul Chill-Out liquid wax (Bio-Rad).    Each sample was incubated at 98C for 2 minutes and 68C for 5 hours.    Five microliters of preheated (68C) 2x DSN Master Buffer (Evrogen) were added to each tube and the mixture incubated at 68C for 10 minutes.    One microliter of DSN enzyme (diluted to between 1U/ul and 1/16U/ul in 1x storage buffer) was added to each sample and incubated at 68C for 25 minutes.    Next 10ul DSN stop solution (Evrogen) was added to each tube followed by a 5 minute 68C incubation.    Finally 40ul water was added and the sample moved to ice to separate from the wax.    The resulting normalized ds cDNA was then cleaned twice on Ampure beads as previously described and eluted in a final volume of 33ul TE.    A modified poly-A tailing and 3' end blocking was then performed on the samples.    First 10.8ul of the normalized ds-cDNA (2-9ng total) was added to 2ul 2.5mM CoCl2 and 2ul 10x TdT buffer and the mixture denatured at 95C for 5 minutes followed by rapid cooling on ice.    One microliter TdT (diluted 1:4, NEB), 4ul 50uM dATP (Roche) and 0.2ul 10mg/ml BSA (NEB) were added and then the samples incubated at 37C for 1 hour and 70C for 10 minutes.    The samples were then denatured at 95C for 5 minutes followed by rapid cooling on ice.    We then added 1ul 10x TdT Buffer, 1ul 2.5mM CoCl2, 0.5ul biotinylated ddATP (Perkin Elmer), 1ul (1:4 diluted ) TdT (NEB) and 6.5ul water and incubated the sample at 37C for 1 hour and 70C for 20 minutes.