TITLE: Enrichment of a specific polyadenylated RNA for nanopore direct RNA sequencing (RNA SPACE)
AUTHORS: Paul MK. Gordon

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This RNA Sequence Picking After Cutting Enzymatically (RNA SPACE) protocol is intended to enrich for a specific polyadenylated RNA, to be performed before the Oxford Nanopore Technologies (ONT) </span><a href="https://store.nanoporetech.com/catalog/product/view/id/297/s/direct-rna-sequencing-kit/category/28/" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">direct RNA sequencing</span></a><span> protocol. This could be used to promote sequencing of a low abundance polyadenylated transcript in a mixture (e.g. polyadenylated viral RNA in a human clinical sample), or to elucidate the unknown 5' of a transcript (i.e. replacement for 5' RACE sequencing). This methods should become increasingly valuable as lower throughput nanopore devices such as the Flongle, Plongle and SmidgION get official support for direct RNA sequencing.</span></div><div class = "text-block"><span>The RNA SPACE protocol takes advantage of the unusual property of six DNA restriction enzymes (</span><a href="https://www.neb.com/products/r0153-avaii" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">AvaII</span></a><span>, </span><a href="https://www.neb.com/products/r0174-avrii" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">AvrII</span></a><span>, </span><a href="https://www.neb.com/products/r0118-bani" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">BanI</span></a><span>, </span><a href="https://www.neb.com/products/r0108-haeiii" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">HaeIII</span></a><span>, </span><a href="https://www.neb.com/products/r0108-haeiii" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">HinfI</span></a><span> and </span><a href="https://www.neb.com/products/r0155-hinfi" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Taq1</span></a><span>) to cut the RNA strand in RNA:DNA duplexes. </span></div><div class = "text-block">This introduces a 3' end that is uniquely targetable using the Oxford Nanopore Technologies protocol's sequence-specific "RTA Oligo B" probe option, rather than the standard poly(dT) overhang version of "RTA Oligo B" which pulls down all polyadenylated transcripts.</div><div class = "text-block">The RNA SPACE software designs two oligonucleotide (oligo) probes for this protocol, for any given gene:</div><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;">the "RE" oligo to generate a RNA:DNA duplex in the known transcripts of interest</li><li style = "counter-reset:ol0;list-style-type:disc;">the sequence-specific RTA Oligo B</li></ul></div></div>

[STEPS]
?. [Oligonucleotide design]
Have the three oligos synthesized (RE oligo, RTA Oligos A & B), by designing your own using the open source software and your reference data.
?. [Enzymatic cleavage of target RNA]
In the same tube, perform 3h or overnight restriction digest according to the NEB protocol for your enzyme, e.g. http://nebcloner.neb.com/#!/protocol/re/single/Taq1 (65C for Taq1, 37C for the other 5 restriction enzymes)The longer incubation is suggested due to the lower efficiency of RNA:DNA restriction compared to DNA:DNA.Step 5 can be performed while you wait for the incubation to finish.
?. [Targeted RNA:DNA duplex formation]
In a 1.5 ml tube, add up to 2µg of your total RNA (in 10µl or less),  and 1µl of 1µM RE oligo.
?. Heat the incubation tube to 80º C for 2 min and let it cool down slowly (0.1º C/sec).
?. [ Sequencing adapter (oligo duplex) formation]
If this is the first time using these custom oligos: In a new tube, anneal standard RTA oligo A and custom RTA oligo B 1:1 at 1.4 µM in buffer (10 mM Tris-HCl pH 7.5, 50 mM NaCl) by heating to 95º C for 2 min and letting them cool down slowly (0.1º C/sec).Otherwise: fetch the custom oligo duplex left over from a previous run.
?. [RNA Purification]
After the 3 hour incubation has finished, perform the RNeasy MinElute Cleanup Kit protocol (spin column) on the contents of the NEB enzyme reaction tube.
?. [Direct RNA Sequencing]
Perform the Oxford Nanopore Technologies Direct RNA sequencing protocol (RNA-002 kit) on the RNA isolated by MinElute. https://community.nanoporetech.com/protocols/ss-direct-rna-sequencing-sqk-rna002/checklist.pdf?devices=minion
?. [Sequencing adapter competitive hybridization]
Add 1ul of the RTA duplex to the tube of eluted RNA. Store any remainder of the RTA duplex, for future uses.
?. [RNA secondary structure reduction]
Incubate 10 minutes at 80C.