TITLE: MPE-seq
AUTHORS: Ben Fair

[STEPS]
?. [RNA fragmentation]
*Fragmentation may eliminate size biases against large primer extensions which are inefficiently PCR amplified and sequenced, but may be unnecessary depending on the application. I usually prefer not to fragment... If you want to do fragmentation, I recommend fragmenting 15 to 40ug RNA by assembling the following reaction in a PCR tube for each RNA sample in PCR tubes. Fragmentation buffer should be added last, and reactions kept on ice to prevent excess fragmentation during reaction set up.  AB1ReagentVolume (uL)215-40ug RNA&nbsp;3Fragmentation buffer24DEPC WaterUp to 205total20
AB1ReagentVolume (uL)215-40ug RNA&nbsp;3Fragmentation buffer24DEPC WaterUp to 205total20
?. [RNA fragmentation]
Induce fragmentation by heating in thermocycler at 65°C for 15 minutes.*(Different RNA samples may need optimization). In past, I have ran a time-course I and decided to use a fragmentation time about half-way between when the rRNA bands start to become indescernable (under fragmented) and saturating (over-fragmented).
?. [RNA fragmentation]
Stop fragmentation by adding 2uL fragmentation STOP buffer and placing samples on ice
?. [Reverse transcription]
For each sample, make a primer/template mix for each sample in a PCR tube. Keep on ice. *Use mastermixes when applicable  AB1ReagentVolume (uL)25X RT buffer43Primer mix (10nM of each gene-specific primer)24Fragmented RNA10ug5WaterUp to 206total20
AB1ReagentVolume (uL)25X RT buffer43Primer mix (10nM of each gene-specific primer)24Fragmented RNA10ug5WaterUp to 206total20
?. [Reverse transcription]
Prepare an enzyme mastermix. Make 21uL per sample in a single PCR tube. If volume exceeds 100uL, split into multiple PCR tubes.  AB1ReagentVolume (uL)25X RT buffer430.1M DTT4410mM dNTP mix25water661mM Biotin dUTP47SSIV18Total21
AB1ReagentVolume (uL)25X RT buffer430.1M DTT4410mM dNTP mix25water661mM Biotin dUTP47SSIV18Total21
?. [Reverse transcription]
Program the following thermocylcer program, preferably on a cycler that can hold temperature during a user-pause 70°C 1min65°C 5min55°C 5min(Optional user pause to hold at 55°C)55°C 30min80°C 10min25°C hold
?. [Reverse transcription]
Add primer mix/template to thermocycler to denature and anneal primers to template. (70°C to 65°C to 55°C)
?. [Reverse transcription]
Once reaction hits 55°C or during user pause, preheat the enzyme mix by placing on thermocycler for a few minutes. Once reaction is preheated, can mix 20uL enzyme mix directly to primer/template mix.*Not preheating enzyme leads to substantial increase of off-target primer-extensions.
?. [Reverse transcription]
Release the user pause and allow the reaction to proceed at 55°C for 30min followed by 80°C inactivation of enzyme.
?. [Reverse transcription]
Degrade the RNA by base hydrolysis:Add 20uL RNA hydrolysis solution. Incubate at 65°C for 15min.
?. [Reverse transcription]
Add 20uL neautralization solution. Sample volume is now 80uL
?. [RNA fragmentation]
Exchange buffer by RNA by ethanol precipitation:Bring sample volume to 50uL by adding 26uL water and transfer to 1.5mL Eppendorf tube. Add 1/10 sample volumes (5uL) 3M sodium acetate, followed by 2.5 sample volumes (137.5uL) of 100% ethanol. Vortex and cool samples to encourage precipitation (2+ hours at -20°C, or 15min at -80°C).
?. [RNA fragmentation]
Centrifuge max-speed in cold centrifuge for 10min to pellet. Pellet should be visible. Aspirate liquid.
?. [RNA fragmentation]
Wash pellet with full sample volume (200uL) of 80% ethanol. Aspirate liquid. Repeat for a total of 2 washes
?. [RNA fragmentation]
Dry pellet as much as possible, by aspirating and air-drying on benchtop before resuspending in water. Aim for a concentration of ~2ug/uL
?. [RNA fragmentation]
Quantify by NanoDrop. Store at -20°C or continue.
?. [Reverse transcription]
Ampure cleanup: Add 1.8 sample volumes AmpureXP beads. Capture beads on magnetic stand for 2min. Aspirate liquidWhile on magnetic stand, add 200uL 70% ethanol wash and incubate for 30 seconds. Aspirate.Repeat for a total of 2 washesRemove from stand and pipet thouroughly with 50uL water to elute.Place on magnetic stand for 2minutes and transfer supernate to labelled 1.5mL centrifuge tube.
?. [First streptavadin bead purification of primer extensions]
Bead preparation:Vortex beads vigorously to resuspendTransfer 20uL of beads per sample to be purified into a new 1.5mL Eppendorf tube (do not add to sample).Place tube on magnetic stand and incubate for 1 minWhile leaving tube on magnetic stand, aspirate bufferRemove tube from magnetic standWash beads by adding 500uL of 1x bind and wash buffer to tube containing the beads and mix well with a pipettePlace tube on magnetic stand and incubate for 1 minAspirate bufferRepeat wash for a total of 2 washesResuspend beads in 50uL of 2x bind and wash buffer per sample to be purifiedMix beads well with a pipetteFor each sample, aliquot 50uL of beads into a new PCR tube or tube for that can fit on magnetic stand.
?. [First streptavadin bead purification of primer extensions]
Bead binding:Add each 50uL prepared beads to each 50uL sample.Place on rotator to gently agitate at RT for 30minPlace tubes on magnetic stand and incubate for 1 minAspirate bufferRemove samples from magnetic stand
?. [First streptavadin bead purification of primer extensions]
Bead wash:Add 150uL of 1x bind and wash buffer and mix well with pipettePlace on magnetic stand and incubate for 1 minaspirate bufferrepeat washing for a total of 3 washes*I think it is acceptable here and in other steps to spray wash volume over beads with the same pipet tip and tap tube to mix if it breaks up the beads.
?. [First streptavadin bead purification of primer extensions]
SSC wash:Wash each sample with 100uL 1x SSC, mixing well by pipetting Place on magnetic stand for 1 minAspirate bufferRemove samples from magnetic stand
?. [First streptavadin bead purification of primer extensions]
Denature/wash off unlabelled strand:Add 100uL of denaturing solution to each sampleIncubate each sample for 10minat RTPlace samples on magnetic stand for 1 minand aspirate bufferWash by adding 100uL of denaturation solution to each sample. Mix well by pipettingPlace samples on magnetic stand for 1 minand aspirate buffer
?. [First streptavadin bead purification of primer extensions]
TE wash:Add 100uL of TE buffer and mix well with pipettePlace on magnetic stand and incubate for 1 minaspirate bufferRepeat TE buffer wash 2 times for a total of 3 washes
?. [First streptavadin bead purification of primer extensions]
Elution:Add 50uL of bead elution buffer to each sampleIncubate samples at 90°Cfor 2 minImmediately place samples on magnetic stand and incubate for 1 minAspirate each sample and transfer to a new tube
?. [First streptavadin bead purification of primer extensions]
Ampure cleanup: Add 1.8 sample volumes AmpureXP beads. Capture beads on magnetic stand for 2min. Aspirate liquidWhile on magnetic stand, add 200uL 70% ethanol wash and incubate for 30 seconds. Aspirate.Repeat for a total of 2 washesRemove from stand and pipet thouroughly with 40uL water to elute.Place on magnetic stand for 2minutes and transfer supernate to labelled 1.5mL centrifuge tube.Store at -20°C or continue.
?. [First strand extension]
AB1ReagentVolume (uL)210X NEB Buffer 25310mM dNTP Mix14cDNA from previous4051stStrand Extension Primer16Total47
AB1ReagentVolume (uL)210X NEB Buffer 25310mM dNTP Mix14cDNA from previous4051stStrand Extension Primer16Total47
?. [First strand extension]
Incubate each sample at 65° Cfor 2 min.
?. [First strand extension]
Cool samples to RT by placing on bench top for~5min.
?. [First strand extension]
Add 3 uL of Klenow exo- enzyme to each sample.
?. [First strand extension]
Incubate samples at RT for 5 min
?. [First strand extension]
Heat samples to 37°C and incubate for 30 min
?. [Second streptavidin bead purification]
Bead preparation:Vortex beads vigorously to resuspendTransfer 20uL of beads per sample to be purified into a new 1.5mL Eppendorf tube (do not add to sample).Place tube on magnetic stand and incubate for 1 minWhile leaving tube on magnetic stand, aspirate bufferRemove tube from magnetic standWash beads by adding 500uL of 1x bind and wash buffer to tube containing the beads and mix well with a pipettePlace tube on magnetic stand and incubate for 1 minAspirate bufferRepeat wash for a total of 2 washesResuspend beads in 50uL of 2x bind and wash buffer per sample to be purifiedMix beads well with a pipetteFor each sample, aliquot 50uL of beads into a new PCR tube or tube for that can fit on magnetic stand.
?. [Second streptavidin bead purification]
Bead binding:Add each 50uL prepared beads to each 50uL sample.Place on rotator to gently agitate at RT for 30minPlace tubes on magnetic stand and incubate for 1 minAspirate bufferRemove samples from magnetic stand
?. [Second streptavidin bead purification]
Bead wash:Add 150uL of 1x bind and wash buffer and mix well with pipettePlace on magnetic stand and incubate for 1 minaspirate bufferrepeat washing for a total of 3 washes*I think it is acceptable here and in other steps to spray wash volume over beads with the same pipet tip and tap tube to mix if it breaks up the beads.
?. [Second streptavidin bead purification]
SSC wash:Wash each sample with 100uL 1x SSC, mixing well by pipetting Place on magnetic stand for 1 minAspirate bufferRemove samples from magnetic stand
?. [Second streptavidin bead purification]
Denature/wash off unlabelled strand:Add 100uL of denaturing solution to each sampleIncubate each sample for 10minat RTPlace samples on magnetic stand for 1 minand aspirate bufferWash by adding 100uL of denaturation solution to each sample. Mix well by pipettingPlace samples on magnetic stand for 1 minand aspirate buffer
?. [Second streptavidin bead purification]
TE wash:Add 100uL of TE buffer and mix well with pipettePlace on magnetic stand and incubate for 1 minaspirate bufferRepeat TE buffer wash 2 times for a total of 3 washes
?. [Second streptavidin bead purification]
Elution:Add 50uL of bead elution buffer to each sampleIncubate samples at 90°Cfor 2 minImmediately place samples on magnetic stand and incubate for 1 minAspirate each sample and transfer to a new tube
?. [Second streptavidin bead purification]
Ampure cleanup: Add 1.8 sample volumes AmpureXP beads. Capture beads on magnetic stand for 2min. Aspirate liquidWhile on magnetic stand, add 200uL 70% ethanol wash and incubate for 30 seconds. Aspirate.Repeat for a total of 2 washesRemove from stand and pipet thouroughly with 20uL water to elute.Place on magnetic stand for 2minutes and transfer supernate to labelled 1.5mL centrifuge tube.Store at -20°C or continue.
?. [PCR amplification and sequencing]
Test PCR:Best to do a test PCR with a small amount of template to verify that it works before using up all of the template!Use NEB 2X HiFi MM with 2uL template  AB1ReagentVolume2Water63template (from previous step)2410uM 7XX Nextera Fwd primer1510uM 5XX Nextera Rev primer16100X SYBR green (disolved in DMSO)0.272X Mastermix108Total20Perform the following qPCR cycling:Denaturation:95 °C  3 minAmplification (40x):98 °C  10 sec62 °C  20 sec72 °C  30 secFinal elongation:72 °C  5 minI typically get Ct values around ~20.The best way to be sure the protocol worked is to look at the qPCR product on a gel, compared to a no template control.
AB1ReagentVolume2Water63template (from previous step)2410uM 7XX Nextera Fwd primer1510uM 5XX Nextera Rev primer16100X SYBR green (disolved in DMSO)0.272X Mastermix108Total20
?. [PCR amplification and sequencing]
Real PCR:Do the same PCR as the test but using all the remaining template in a 40uL reaction and only do as many PCR cycles as necessary to get amplification. Clean with Ampure beads.
?. [PCR amplification and sequencing]
Sequence. MPE-seq libraries from human samples are not very complex in my experience. Perhaps on the order of a million or couple million unique molecules (UMIs) so there is diminishing returns with more sequencing. I think 1-5 million reads is usually sufficient. I usually observe 20-200 fold enrichment of targets compared to RNA-seq, though it varies quite a bit from target to target.Unlike the MPE-seq publication in yeast, for whatever reason, we have not reads that appear derived from lariat intermediates, though we can measure plenty of unspliced message. In yeast, we reported ~ a 1:10 ratio of lariat derived reads to fully unspliced reads. That ratio is likely much less for humans.