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.    Between 100-200ng of RNA was used for each cDNA synthesis reaction using the Superscript III kit (Invitrogen).    Each sample of RNA was resuspended in 17ul DEPC water and incubated at 95C for 5 minutes then rapidly cooled on ice.    First, 10ul of 50ng/ul random hexamers and 2ul 10mM dNTP mix were added.    The mixture was incubated at 65C for 5 minutes then rapidly cooled on ice.    Next, 5ul 10x buffer, 1ul 0.1M DTT and 10ul 25mM MgCl2 were added and the mixture incubated at 15C for 20 minutes.    Then 2.5ul RnaseOut and 2.5ul Superscript III were added and the samples incubated at 25C for 10 minutes, 40C for 40 minutes, 55C for 50 minutes and 85C for 5 minutes.    Upon completion of cDNA synthesis RNA was removed by the addition of 1ul RnaseH and 1ul RNAseIf (NEB) and incubation at 37C for 30 minutes.    The resulting cDNA was then purified by the serial use of two Performa Gel Filtration Columns (EdgeBio) and quantified.    Using 100ng of the prepared cDNA in 28ul water, 5ul Helicos PolyA Control Oligo was added and the mixture incubated at 95C for 5 minutes followed by rapid cooling on ice.    A mixture of 5ul 2.5mM CoCl2, 5ul 10X Terminal deoxynucleotide Transfer (TdT) buffer, 5ul Helicos PolyA tailing dATP and 5ul TdT was then added to the sample and incubated at 42C for 1 hour and 70C for 10 minutes.    The sample was denatured at 95C for 5 minutes then rapidly cooled on ice.    We then added 0.4ul biotinylated ddATP (Perkin Elmer) and 2ul TdT (NEB) and incubated the sample at 37C for 1 hour and 70C for 10 minutes to block the 3' tail of the cDNA.    The sample was then digested with 1ul USER enzyme (NEB) and incubated at 37C for 30 minutes to remove the control oligo.    The sample was then purified using AMPure beads (Beckman Coulter) by bringing the volume up to 60ul and mixing with 72ul AMPure beads and incubating the mixture at room temperature for 30 minutes with intermittent agitation.    Using a magnetic stand the beads were then collected and washed twice with 500ul 70% ethanol.    The beads were allowed to dry for 5-10 minutes, resuspended in 20ul TE buffer and left on the stand for 5 minutes before the supernatant was removed.    An additional 20ul elution was performed and pooled with the first.