TITLE: A rapid, sensitive, scalable method for Precision Run-On sequencing (qPRO-seq)
AUTHORS: Julius Judd, Luke A. Wojenski, Lauren M. Wainman, Nathaniel D. Tippens, Edward J. Rice, Alexis Dziubek, Geno J. Villafano, Erin M. Wissink, Philip Versluis, Lina Bagepalli, Sagar R. Shah, Dig B. Mahat, Jacob M. Tome, Charles G. Danko, John T. Lis, Leighton J. Core

[DESCRIPTION]
<div class = "text-blocks"></div>

[STEPS]
?. [Cell Permeabilization]
Prepare permeabilization buffer, wash buffer, and freeze buffer and place .
on ice
CAUTION: DEPC is toxic and harmful!
CRITICAL: Care should be taken to avoid nuclease contamination. Change gloves routinely and prepare/use nuclease-free reagents.
CRITICAL: ALL steps should be carried out on ice or in a cold room
All salt solutions should be prepared in ddH2O. Then add 0.1% (v/v) DEPC, stir overnight, and autoclave. Tris buffers instead need to be carefully prepared with DEPC-treated ddH2O.
All other solutions (detergents, DTT, sucrose, EDTA/EGTA, and Tris buffers) should be prepared in DEPC ddH2O in RNase free containers and filter sterilized. Glassware can be made RNase by filling with water, adding 0.1% (v/v) DEPC, incubating with agitation overnight, and autoclaving. Alternatively, glassware can be baked at 300 °C for 4 hours.
The permeabilization buffer, cell wash buffer, freeze buffer, and bead washing/binding buffers can be made and filter-sterilized in advance without the DTT, SUPERase-In™ RNase Inhibitor, and Pierce™ protease inhibitor tablets. DTT, SUPERase-In™ RNase Inhibitor, and protease inhibitor tablets can be added when buffers are needed. Store buffers at 4°C. Use DEPC treated glassware or RNase free plasticware.
?. [Cell Permeabilization]
Permeabilization Buffer:  Tris-Cl, pH 8.0 KCl SucroseMgCl2 EGTA IgepalDTT Tween-20 Glycerol in DEPC-treated ddH2O. Add 1 Pierce protease inhibitor tablet and  SUPERase-In RNase inhibitor per .
10 µl
50 ml
?. [Cell Permeabilization]
Cell Wash buffer:  Tris-Cl, pH 8.0 KClsucrose MgCl2 EGTA DTT Glycerol in DEPC-treated ddH2O. Add 1 Pierce protease Inhibitor tablet and  SUPERase-In RNase inhibitor per .
10 µl
50 ml
?. [Cell Permeabilization]
Freeze buffer:  Tris-Cl, pH 8.0 glycerol MgCl2 EDTA DTT in DEPC-treated ddH2O. Add  SUPERase-In RNase inhibitor per .
10 µl
50 ml
?. [Cell Permeabilization]
Proceed using one of the following options:
?. [Cell Permeabilization]
Option 2.1.: Adherent cells (volumes are for 10 cm plates):2.1.1. Wash cells with  ice cold PBS.2.1.2. Repeat the PBS wash step for a total of two washes.2.1.3. Add  ice cold permeabilization buffer, scrape cells, and transfer to a conical tube.2.1.4. Rinse plate with  permeabilization buffer and pool cells in conical tube (Vf = ).
10 ml
5 ml
5 ml
10 ml
?. [Cell Permeabilization]
Option 2.2: Suspension cells:2.2.1. Transfer cells into conical tubes and spin down at 700–1000 x g for  at .2.2.2. Wash with  ice cold PBS.2.2.3. Repeat the PBS wash for a total of two washes.2.2.4. Resuspend in  cold permeabilization buffer.
4 °C
10 ml
10 ml
Use a centrifuge with a swinging bucket rotor for all centrifuge steps during cell permeabilization. Using a fixed angle rotor will shear cells, releasing a smear of white chromatin.
Centrifuge speed is cell size dependent. We typically centrifuge HeLa at 800 x g and Drosophila at 1,000 x g.
When resuspending cells during permeabilization after centrifugation steps, first gently resuspend the cell pellet with 1 mL solution with a wide-bore P1000 tip. Then add the remaining volume (usually 9 mL) and mix by gentle inversion.
?. [Cell Permeabilization]
Incubate on ice for .
?. [Cell Permeabilization]
Check for permeabilization with Trypan blue. Greater than 98% permeabilization is ideal.
If your cell type is not permeabilized under these conditions, add Triton X-100 to  — .
?. [Cell Permeabilization]
Spin down at 700–1000 x g for  at .
4 °C
Centrifuge speed is cell size dependent. We typically centrifuge HeLa at 800 x g and Drosophila at 1,000 x g.
?. [Cell Permeabilization]
Wash with 10 mL ice cold cell wash buffer.
When resuspending cells during permeabilization after centrifugation steps, first gently resuspend the cell pellet with 1 mL solution with a wide-bore P1000 tip. Then add the remaining volume (usually 9 mL) and mix by gentle inversion.
?. [Cell Permeabilization]
Repeat the cell wash buffer wash for a total of two washes.
?. [Cell Permeabilization]
Decant wash buffer, and then carefully pipette off remaining buffer and discard without disturbing the cell pellet.
?. [Cell Permeabilization]
Using wide-bore tips, resuspend in  cold freeze buffer and transfer to a 1.5 mL tube.
250 µl
?. [Cell Permeabilization]
Rinse the conical tube with an additional  freeze buffer and pool (Vf = ).
250 µl
500 µl
?. [Cell Permeabilization]
Count cells and add permeabilized spike-in cells if desired.
When processing multiple samples, if counting will cause the cells to sit on ice for greater than 10 min, reserve 10 µL for counting, aliquot cells in 100 µL aliquots, and snap freeze. Count the cells and then adjust the concentration with freeze buffer after thawing and prior to the run-on.
In order to robustly normalize between conditions where a dramatic change in global transcription levels are expected, we add a fixed number of cells of a different species to a fixed number of experimental cells at the permeabilization step. Reads can be mapped to a combined genome, and the number of spike-in mapped reads can then be used as a scaling factor. These cells should be permeabilized prior to the experiment, aliquoted, and added to 1-2% by cell number after permeabilization and counting, either just prior to freezing or just prior to the run-on reaction. We frequently use Drosophila S2 cells to normalize human cell experiments and vice versa.
?. [Cell Permeabilization]
Spin down at 1000 x g for  at .
4 °C
Microfuge tubes can be spun in a fixed angle rotor, but we continue to use a swinging bucket rotor so that cells collect at bottom of tube (this tends to decrease cell loss).
?. [Cell Permeabilization]
Resuspend the desired number of cells for each run-on reaction in  freeze buffer.
52 µl
We have had success performing this protocol with as few as 50k primary human cells. In general, we find that the quality of libraries will increase until ~1 x 106 cells per run-on but using more cells than this offers little benefit. This will also depend on how transcriptionally active a given cell type is and genome size.
?. [Cell Permeabilization]
Continue to the run-on or snap freeze  aliquots in LN2 and store at .
52 µl
-80 °C
Permeabilized cells are stable indefinitely at -80°C (Chu et al., 2018).
?. [Preparation for the Run-On]
Pre-chill a microcentrifuge to .
4 °C
?. [Preparation for the Run-On]
Set a heat block with water in the wells to  and another to  and allow temperature to equilibrate.
37 °C
65 °C
A thermomixer set to 37 °C can also be used for incubating the run-on reactions.
?. [Preparation for the Run-On]
Prepare bead preparation buffer, high salt wash buffer, low salt wash buffer, and binding buffer, and store
on ice
The permeabilization buffer, cell wash buffer, freeze buffer, and bead washing/binding buffers can be made and filter-sterilized in advance without the DTT, SUPERase-In™ RNase Inhibitor, and Pierce™ protease inhibitor tablets. DTT, SUPERase-In™ RNase Inhibitor, and protease inhibitor tablets can be added when buffers are needed. Store buffers at 4°C. Use DEPC treated glassware or RNase free plasticware.
?. [Preparation for the Run-On]
For each run-on reaction, wash  Dynabeads™ MyOne™ Streptavidin C1 Beads once in  bead preparation buffer using a magnet stand. Beads can be washed in bulk.
10 µl
1 ml
CRITICAL:Be sure to properly resuspend beads prior to aliquoting them.
C1 Streptavidin beads are preferred compared to M280 beads because they have higher binding capacity and use a negatively charged matrix. This significantly reduces carryover of non-biotinylated RNAs including adapter dimers. Be careful not to disturb beads when removing buffers from tubes. Open tube caps prior to placing them on the magnet stand, as opening on the magnet stand can disturb the liquid. Check pipette tip against a white background before discarding liquid to ensure beads are not present.
?. [Preparation for the Run-On]
Wash beads twice with  binding buffer.
1 ml
Always quickly spin samples down using a picofuge to remove liquid from tube caps between washes.
?. [Preparation for the Run-On]
Resuspend the beads in  binding buffer per sample. Place beads  or at  until needed.
25 µl
on ice
4 °C
?. [Preparation for the Run-On]
Bead Preparation Buffer: NaOH NaCl in DEPC-treated ddH2O.
?. [Preparation for the Run-On]
Bead Binding Buffer: Tris-HCl, pH 7.4 NaCl Triton X-100 EDTA in DEPC-treated ddH2O. Add  SUPERase-In RNase Inhibitor per .
2 µl
10 ml
?. [Preparation for the Run-On]
Low Salt Wash Buffer: Tris-HCl, pH 7.4Triton X-100EDTAin DEPC-treated ddH2O. Add  SUPERase-In RNase Inhibitor per .
2 µl
10 ml
?. [Preparation for the Run-On]
High Salt Wash buffer:  Tris-HCl, pH 7.4 NaCl Triton X-100 EDTAin DEPC-treat H2O. Add  SUPERase-In RNase Inhibitor per .
2 µl
10 ml
?. [Run-On Reaction]
Prepare 2XROMM equilibrate at  ( for Drosophila).
37 °C
30 °C
When preparing the 2XROMM, first add all components other than Sarkosyl and mix by vortexing on high for >10 sec. Collect the solution with a quick spin, add Sarkosyl, and mix thoroughly by pipetting carefully to avoid bubbles. If you leave the 2XROMM on ice, a precipitate can form. Before use, check if this has occurred. The precipitate can be re-dissolved by heating at 37 °C for ~5 min and pipette mixing.
?. [Run-On Reaction]
Using a wide bore tip, add of permeabilized cells to new 1.5 mL tube.
50 µl
?. [Run-On Reaction]
Pipette  of preheated 2XROMM into each reaction tube (already containing permeabilized cells). Gently and thoroughly pipette the mixture 15 times.
50 µl
It is extremely important to thoroughly mix the reaction so that nucleotides diffuse into highly viscous chromatin!
?. [Run-On Reaction]
Incubate in a heat block or thermomixer at  ( for Drosophila) at 750 RPM for . Have RL buffer from Norgen kit or TRIzol LS ready for use.
37 °C
30 °C
?. [Run-On Reaction]
Proceed to step 26.1 or 26.2 depending on choice of RNA extraction method immediately after the reaction is complete (take the sample off the heat block and immediately add buffer RL or TRIzol LS).
Stagger both addition of the 2XROMM and addition of TRIzol or buffer RL by 30 seconds or 1 minute, to ensure each sample is incubated exactly 5 min.
?. [Run-On Reaction]
2X Run-On Master Mix (2XROMM):  Tris-Cl, pH 8.0MgCl2 DTT KCl Biotin-11-CTP Biotin-11-UTP Biotin-11-ATP Biotin-11-GTP Sarkosylin DEPC-treated ddH2OAdd  SUPERase-In RNase Inhibitor per reaction.
1 µl
The run-on reaction uses 4 biotin-NTPs. However, ATP and GTP can be substituted at equal concentration for Biotin-11-ATP and Biotin-11-GTP to reduce cost. Biotin-11-ATP and biotin-11-GTP are 10X as expensive as biotin-11-CTP and biotin-11-UTP. With two biotin-NTPs blocking elongation, each polymerase can be expected to extend ~5 nt or less which we find gives sufficient resolution for the vast majority of applications. For low cell number experiments, increase the concentration of the biotin-NTPs to 500 µM. Biotin-NTP incorporation efficiency is ~60% with the concentration in the 2XROMM as written, which is sufficient for experiments using 106cells or greater, but increasing the concentration improves incorporation to ~77%.
?. [Total RNA extraction and Base Hydrolysis]
Proceed from Step 25 to one of the following options:
TRIzol LS or the Norgen Total RNA Purification Kit can be used to extract total RNA from the run-on reaction. Both options produce identical results. The Norgen kit is faster and less technically challenging to use, but more expensive. If TRIzol LS is used, Micro Bio-Spin™ RNase free P-30 Gel Columns are also needed to remove unincorporated biotin-NTPs as the biotin concentration will otherwise overwhelm the binding capacity of the streptavidin beads.
?. [Total RNA extraction and Base Hydrolysis]
Option 26.1: NORGEN RNA Extraction:1. Add  RL buffer and vortex.2. Add  ethanol and vortex.3. Apply solution to Norgen RNA extraction column.4. Spin at 3,500 x g for  at .5. Add  wash solution A (ensure ethanol has been added).6. Spin at 14,000 x g for  at .7. Discard flow through.8. Repeat wash (steps 6 & 7) for a total of two washes.9. Spin at 14,000 x g for  to dry column.10. Add  DEPC-treated ddH2O and vortex.11. Elute by spinning at 200 x g for  at  and then at 14,000 x g for  at .12. Elute again with  DEPC-treated ddH2O and pool eluates (Vf = ).13. Denature at  for  and then snap cool on ice.14. Add  ice cold  NaOH and incubate  on ice. 15. Add  cold  Tris-Cl pH 6.8, mix by pipetting.16. Add NaCl and  GlycoBlue and mix.17. Add  Ethanol and vortex.18. Centrifuge the samples at >20,000 x g for  at .19. Carefully pipette supernatant off and discard.20. Add  ethanol.21. Mix by gentle inversion and spin down briefly.22. Carefully pipette supernatant off and discard.23. Airdry the RNA pellet.24. Resuspend in  DEPC-treated ddH2O.
350 µl
240 µl
25 °C
400 µl
25 °C
50 µl
25 °C
25 °C
50 µl
100 µl
65 °C
25 µl
125 µl
5 µl
1 µl
625 µl
If the protocol needs to be performed over two days, the ethanol precipitation in step 26.1.17 is the safest overnight stopping point. Store samples at -80 °C.
4 °C
A blue pellet should be visible at the bottom of tube. The pellet can be difficult to see but should be visible. It may appear spread out. If a pellet is not visible, vortex well and repeat spin.
When removing the supernatant before the 70% ethanol wash be careful not to disturb the pellet. Approximately 30–50μL of ethanol can be left in the tube to avoid disturbing the pellet prior to adding the 70% ethanol wash. This procedure can also be used after the 70% ethanol wash (step 25.1.22), but then remove the final 30-50 µL using a P200 tip after a quick spin in a picofuge.
750 µl
Air dry the RNA pellet by leaving tubes open in fume hood to prevent contamination. This will take ~3-10 min depending on how much ethanol is left in the tube. Do not to let the RNA pellet dry completely as this will greatly decrease its solubility.
6 µl
?. [Total RNA extraction and Base Hydrolysis]
Option 26.2: Trizol LS RNA Extraction:1. Add  TRIzol LS with a wide bore P1000 tip and carefully pipette >10X until all white globs of nucleoproteins are homogenized. 2. Pipette mix again with a standard bore P1000 tip. Samples should be completely homogenous.3. Vortex vigorously for at least .4. Incubate samples on ice until all run-on reactions are complete.5. Add  chloroform.6. Vortex the samples at max speed for , then incubate on ice for .7. Centrifuge the samples at >20,000 x g for  at .8. Transfer the ~ aqueous phase into a new tube.9. Add  of GlycoBlue and mix.10. Add 2.5X volumes (~)  ethanol and vortex. 11. Centrifuge at > 20,000 x g for  at .12. Carefully pipette supernatant off and discard.13. Add  ethanol.14. Mix by gentle inversion and quickly spin down.15. Carefully pipette supernatant off and discard.16. Airdry the RNA pellet.17. Resuspend in  DEPC-treated ddH2O.18. Briefly denature at  for  and then snap cool .19. Add  ice cold NaOH and incubate  for . 20. Add  Tris-Cl , mix by pipetting.21. Pass through a calibrated Bio-Rad RNase free P-30 column (follow manufacturer’s instructions).22. Bring volume to  with DEPC-treated ddH2O (add ~ ).23. Add  Glycoblue and  NaCl and vortex.24. Add  ethanol and vortex.25. Centrifuge at >20,000 x g for  at .26. Carefully pipette supernatant off and discard.27. Add  ethanol.28. Mix by gentle inversion and quickly spin down.29. Carefully pipette supernatant off and discard.30. Airdry the RNA pellet.31. Resuspend in  DEPC-treated ddH2O.
250 µl
65 µl
When pipetting chloroform, always pipette twice because the first draw always leaks.
4 °C
200 µl
When transferring the aqueous phase of TRIzol extractions to a new tube, tilt the tube to a 45° angle and carefully remove only the clear liquid. Avoid contamination by the pink organic phase or white interphase.
1 µl
500 µl
4 °C
A blue pellet should be visible at the bottom of tube. The pellet can be difficult to see but should be visible. It may appear spread out. If a pellet is not visible, vortex well and repeat spin.
When removing the supernatant before the 70% ethanol wash be careful not to disturb the pellet. Approximately 30–50μL of ethanol can be left in the tube to avoid disturbing the pellet prior to adding the 70% ethanol wash. This procedure can also be used after the 70% ethanol wash (step 26.2.15), but then remove the final 30-50 µL using a P200 tip after a quick spin in a picofuge.
750 µl
Air dry the RNA pellet by leaving tubes open in fume hood to prevent contamination. This will take ~3-10 min depending on how much ethanol is left in the tube. Do not to let the RNA pellet dry completely as this will greatly decrease its solubility.
30 µl
65 °C
on ice
7.5 µl
on ice
37.5 µl
200 µl
125 µl
1 µl
8 µl
500 µl
If the protocol needs to be performed over two days, the ethanol precipitation in 26.2.24 is the safest overnight stopping point. Store samples at -80 °C.
4 °C
A blue pellet should be visible at the bottom of tube. The pellet can be difficult to see but should be visible. It may appear spread out. If a pellet is not visible, vortex well and repeat spin.
When removing the supernatant before the 70% ethanol wash be careful not to disturb the pellet. Approximately 30–50μL of ethanol can be left in the tube to avoid disturbing the pellet prior to adding the 70% ethanol wash. This procedure can also be used after the 70% ethanol wash (step 26.2.29), but then remove the final 30-50 µL using a P200 tip after a quick spin in a picofuge.
750 µl
Air dry the RNA pellet by leaving tubes open in fume hood to prevent contamination. This will take ~3-10 min depending on how much ethanol is left in the tube. Do not to let the RNA pellet dry completely as this will greatly decrease its solubility.
6 µl
?. [3'RNA Adapter Ligation]
Continue here from step 26.1.24 or 26.2.31:
?. [3'RNA Adapter Ligation]
Add  VRA3 (Vf = ).
1 µl
7 µl
The concentration of RNA adapters in the ligation steps (1 µL 10 µM) is optimal for approximately 106 mammalian cells. For lower cell numbers, the adapter concentration must be diluted to limit dimer formation. We dilute linearly with cell concentration relative to this established concentration, i.e. 1 µL 5 µM for 5 x 105 cells, 1 µL 2.5 µM for 2.5 x 105 cells, etc.
?. [3'RNA Adapter Ligation]
Denature at  for  and snap cool .
65 °C
on ice
?. [3'RNA Adapter Ligation]
Prepare ligation mix in the following order:  AB1ReagentVolume210X T4 RNA Ligase Buffer2 µL3ATP (10 mM)2 µL4SUPERase-In RNase Inhibitor1 µL550% PEG80006 µL6T4 RNA LIgase 1 (ssRNA ligase)2 µL
AB1ReagentVolume210X T4 RNA Ligase Buffer2 µL3ATP (10 mM)2 µL4SUPERase-In RNase Inhibitor1 µL550% PEG80006 µL6T4 RNA LIgase 1 (ssRNA ligase)2 µL
Pipette slowly because 50% PEG8000 is very viscous. Heating 50% PEG8000 makes it easier to pipette. Pipette the ligation mix until it is homogenous before use.
When preparing enzymatic reaction mixtures that contain SUPERase-In RNase Inhibitor, a fixed volume (1 µL) SUPERase-In can be added to the entire master mix regardless of number of reactions to decrease cost. Bring the remainder of the master mix up to the required volume with DEPC-treated ddH2O. Murine RNase inhibitor can also be substituted to limit costs for all steps after the run-on. SUPERase-In is recommended prior to the run-on as it inhibits T1 RNase.
?. [3'RNA Adapter Ligation]
Add  and mix by pipetting 10–15X (Vf = ).
13 µl
20 µl
?. [3'RNA Adapter Ligation]
Incubate at for .
25 °C
?. [Streptavidin Bead Binding]
Add  binding buffer to each sample (Vf = ).
55 µl
75 µl
?. [Streptavidin Bead Binding]
Add  pre-washed beads to each sample (Vf = ).
25 µl
100 µl
?. [Streptavidin Bead Binding]
Incubate for at  with end to end rotation.
25 °C
?. [Streptavidin Bead Binding]
Wash once with  High Salt Wash buffer with tube swap.
500 µl
Be careful not to disturb beads when removing buffers from tubes. Open tube caps prior to placing them on the magnet stand, as opening on the magnet stand can disturb the liquid. Check pipette tip against a white background before discarding liquid to ensure beads are not present.
For each washing step gently invert tubes 10–15X, quickly spin down with a picofuge, open caps, and then place on the magnet stand. Wait 1-2 minutes and pipette the supernatant off without disturbing the beads. If there are bubbles in the tube carefully pipette them off first and then remove supernatant. Beware that bubbles may dislodge beads from the side of the tube. After removing the bulk of the liquid, collect remaining liquid with a quick spin in a picofuge, place the tube back on the magnet stand, and carefully remove remaining liquid by pipetting.
Transferring beads to a new tube after the binding incubation—during the high salt wash step—helps limit adapter dimer formation. After resuspending the beads in High Salt buffer, quickly spin down with a picofuge, resuspend beads by gently pipetting, and carefully transfer to a new tube. Pipette slowly to avoid bead loss! Place this new tube on the magnet stand and proceed with the washing protocol.
?. [Streptavidin Bead Binding]
Wash once with  Low Salt Wash buffer.
500 µl
Be careful not to disturb beads when removing buffers from tubes. Open tube caps prior to placing them on the magnet stand, as opening on the magnet stand can disturb the liquid. Check pipette tip against a white background before discarding liquid to ensure beads are not present.
For each washing step gently invert tubes 10–15X, quickly spin down with a picofuge, open caps, and then place on the magnet stand. Wait 1-2 minutes and pipette the supernatant off without disturbing the beads. If there are bubbles in the tube carefully pipette them off first and then remove supernatant. Beware that bubbles may dislodge beads from the side of the tube. After removing the bulk of the liquid, collect remaining liquid with a quick spin in a picofuge, place the tube back on the magnet stand, and carefully remove remaining liquid by pipetting.
Do not allow streptavidin beads to dry completely, as this can lead to clumping and make full resuspension impossible. When processing multiple samples, remove liquid from the previous wash or enzymatic step from the first sample and immediately resuspend those beads in the next solution, then repeat this process for additional samples.
?. [On-Bead 5’ Hydroxyl Repair]
Resuspend beads in  PNK mix (Vf = :  AB1ReagentVolume2DEPC-treated ddH2O13 µL310X PNK buffer2 µL410 mM ATP2 µL5T4 Polynucleotice Kinase1 µL6SUPERase-In RNase Inhibitor1 µL
19 µl
20 µl
AB1ReagentVolume2DEPC-treated ddH2O13 µL310X PNK buffer2 µL410 mM ATP2 µL5T4 Polynucleotice Kinase1 µL6SUPERase-In RNase Inhibitor1 µL
On-bead reaction volumes assume that 1 µL of liquid remains on the beads.
When preparing enzymatic reaction mixtures that contain SUPERase-In RNase Inhibitor, a fixed volume (1 µL) SUPERase-In can be added to the entire master mix regardless of number of reactions to decrease cost. Bring the remainder of the master mix up to the required volume with DEPC-treated ddH2O. Murine RNase inhibitor can also be substituted to limit costs for all steps after the run-on. SUPERase-In is recommended prior to the run-on as it inhibits T1 RNase.
?. [On-Bead 5’ Decapping]
Place the tubes on a magnet stand and remove supernatant.
Be careful not to disturb beads when removing buffers from tubes. Open tube caps prior to placing them on the magnet stand, as opening on the magnet stand can disturb the liquid. Check pipette tip against a white background before discarding liquid to ensure beads are not present.
Always quickly spin samples down using a picofuge to remove liquid from tube caps.
Do not allow streptavidin beads to dry completely, as this can lead to clumping and make full resuspension impossible. When processing multiple samples, remove liquid from the previous wash or enzymatic step from the first sample and immediately resuspend those beads in the next solution, then repeat this process for additional samples.
?. [On-Bead 5’ Decapping]
Resuspend the beads in  RppH mix (Vf = ):  AB1ReagentVolume2DEPC-treated ddH2O15 µL310X ThermoPol Buffer2 µL4RppH1 µL5SUPERase-In RNase Inhibitor1 µL
19 µl
20 µl
AB1ReagentVolume2DEPC-treated ddH2O15 µL310X ThermoPol Buffer2 µL4RppH1 µL5SUPERase-In RNase Inhibitor1 µL
On-bead reaction volumes assume that 1 µL of liquid remains on the beads.
We have also successfully used Cap-Clip™ Acid Pyrophosphatase (CELLTREAT) instead of RppH. Cap-Clip has lower buffer pH which may alleviate base hydrolysis of RNA that could occur in the pH 8.0 ThermoPol buffer. However, this is not a major concern except for in the most sensitive of applications.
When preparing enzymatic reaction mixtures that contain SUPERase-In RNase Inhibitor, a fixed volume (1 µL) SUPERase-In can be added to the entire master mix regardless of number of reactions to decrease cost. Bring the remainder of the master mix up to the required volume with DEPC H2O. Murine RNase inhibitor can also be substituted to limit costs for all steps after the run-on. SUPERase-In is recommended prior to the run-on as it inhibits T1 RNase.
?. [On-Bead 5’ Decapping]
Incubate at  for.
37 °C
Mix on-bead reactions by gently flicking the tubes every 10 minutes.
?. [On-Bead 5’ Hydroxyl Repair]
Incubate at  for.
37 °C
Mix on-bead reactions by gently flicking the tubes every 10 minutes.
?. [On-Bead 5’ RNA Adaptor Ligation]
Place the tubes on a magnet stand and remove supernatant.
Be careful not to disturb beads when removing buffers from tubes. Open tube caps prior to placing them on the magnet stand, as opening on the magnet stand can disturb the liquid. Check pipette tip against a white background before discarding liquid to ensure beads are not present.
Always quickly spin samples down using a picofuge to remove liquid from tube caps.
Do not allow streptavidin beads to dry completely, as this can lead to clumping and make full resuspension impossible. When processing multiple samples, remove liquid from the previous wash or enzymatic step from the first sample and immediately resuspend those beads in the next solution, then repeat this process for additional samples.
?. [On-Bead 5’ RNA Adaptor Ligation]
Resuspend the beads in  adapter mix (Vf = ):  AB1ReagentVolume2DEPC-treated ddH2O6 µL3REV5 (10 µM)1 µl
7 µl
8 µl
AB1ReagentVolume2DEPC-treated ddH2O6 µL3REV5 (10 µM)1 µl
The concentration of RNA adapters in the ligation steps (1 µL 10 µM) is optimal for approximately 106 mammalian cells. For lower cell numbers, the adapter concentration must be diluted to limit dimer formation. We dilute linearly with cell concentration relative to this established concentration, i.e. 1 µL 5 µM for 5 x 105 cells, 1 µL 2.5 µM for 2.5 x 105 cells, etc.
?. [On-Bead 5’ RNA Adaptor Ligation]
Denature at  for , then snap cool .
65 °C
on ice
?. [On-Bead 5’ RNA Adaptor Ligation]
Prepare ligation mix in the following order:  AB1ReagentVolume210X T4 RNA ligase buffer2 µL3ATP (10 mM)2 µL4SUPERase-In RNase Inhibitor1 µL550% PEG80006 µL6T4 RNA Ligase 1 (ssRNA ligase)1 µL
AB1ReagentVolume210X T4 RNA ligase buffer2 µL3ATP (10 mM)2 µL4SUPERase-In RNase Inhibitor1 µL550% PEG80006 µL6T4 RNA Ligase 1 (ssRNA ligase)1 µL
Pipette slowly because 50% PEG8000 is very viscous. Heating 50% PEG8000 makes it easier to pipette. Pipette the ligation mix until it is homogenous before use.
Pipette slowly because 50% PEG8000 is very viscous. Heating 50% PEG8000 makes it easier to pipette. Pipette the ligation mix until it is homogenous before use.
On-bead reaction volumes assume that 1 µL of liquid remains on the beads.
?. [On-Bead 5’ RNA Adaptor Ligation]
Add  to each tube (Vf = ).
12 µl
20 µl
?. [On-Bead 5’ RNA Adaptor Ligation]
Incubate at for .
25 °C
Mix on-bead reactions by gently flicking the tubes every 10 minutes.
?. [TRIzol Elution of RNA]
Wash once with  High Salt Wash buffer with tube swap.
500 µl
Be careful not to disturb beads when removing buffers from tubes. Open tube caps prior to placing them on the magnet stand, as opening on the magnet stand can disturb the liquid. Check pipette tip against a white background before discarding liquid to ensure beads are not present.
For each washing step gently invert tubes 10–15X, quickly spin down with a picofuge, open caps, and then place on the magnet stand. Wait 1-2 minutes and pipette the supernatant off without disturbing the beads. If there are bubbles in the tube carefully pipette them off first and then remove supernatant. Beware that bubbles may dislodge beads from the side of the tube. After removing the bulk of the liquid, collect remaining liquid with a quick spin in a picofuge, place the tube back on the magnet stand, and carefully remove remaining liquid by pipetting.
Transferring beads to a new tube after the binding incubation—during the high salt wash step—helps limit adapter dimer formation. After resuspending the beads in High Salt buffer, quickly spin down with a picofuge, resuspend beads by gently pipetting, and carefully transfer to a new tube. Pipette slowly to avoid bead loss! Place this new tube on the magnet stand and proceed with the washing protocol.
?. [TRIzol Elution of RNA]
Wash once with  Low Salt Wash buffer.
500 µl
Be careful not to disturb beads when removing buffers from tubes. Open tube caps prior to placing them on the magnet stand, as opening on the magnet stand can disturb the liquid. Check pipette tip against a white background before discarding liquid to ensure beads are not present.
For each washing step gently invert tubes 10–15X, quickly spin down with a picofuge, open caps, and then place on the magnet stand. Wait 1-2 minutes and pipette the supernatant off without disturbing the beads. If there are bubbles in the tube carefully pipette them off first and then remove supernatant. Beware that bubbles may dislodge beads from the side of the tube. After removing the bulk of the liquid, collect remaining liquid with a quick spin in a picofuge, place the tube back on the magnet stand, and carefully remove remaining liquid by pipetting.
Do not allow streptavidin beads to dry completely, as this can lead to clumping and make full resuspension impossible. When processing multiple samples, remove liquid from the previous wash or enzymatic step from the first sample and immediately resuspend those beads in the next solution, then repeat this process for additional samples.
?. [TRIzol Elution of RNA]
Resuspend beads in  TRIzol.
300 µl
?. [TRIzol Elution of RNA]
Vortex at max speed for > , then incubate  for .
on ice
?. [TRIzol Elution of RNA]
Add  chloroform.
60 µl
When pipetting chloroform, always pipette twice because the first draw always leaks.
?. [TRIzol Elution of RNA]
Vortex at max speed for , then incubate  for .
on ice
?. [TRIzol Elution of RNA]
Centrifuge at > 20,000 x g for  at .
4 °C
?. [TRIzol Elution of RNA]
Transfer the aqueous phase (~) to a new tube.
180 µl
When transferring the aqueous phase of TRIzol extractions to a new tube, tilt the tube to a 45° angle and carefully remove only the clear liquid. Avoid contamination by the pink organic phase or white interphase.
?. [TRIzol Elution of RNA]
Add  GlycoBlue and mix.
1 µl
?. [TRIzol Elution of RNA]
Add 2.5X volumes (~)  ethanol and vortex.
450 µl
?. [TRIzol Elution of RNA]
Centrifuge the samples at > 20,000 x g for  at  .
4 °C
A blue pellet should be visible at the bottom of tube. The pellet can be difficult to see but should be visible. It may appear spread out. If a pellet is not visible, vortex well and repeat spin.
?. [TRIzol Elution of RNA]
Carefully pipette supernatant off and discard.
When removing the supernatant before the 70% ethanol wash be careful not to disturb the pellet. Approximately 30–50μL of ethanol can be left in the tube to avoid disturbing the pellet prior to adding the 70% ethanol wash. This procedure can also be used after the 70% ethanol wash (step 63), but then remove the final 30-50 µL using a P200 tip after a quick spin in a picofuge.
?. [TRIzol Elution of RNA]
Add  ethanol.
750 µl
?. [TRIzol Elution of RNA]
Mix by gentle inversion and quickly spin down.
?. [TRIzol Elution of RNA]
Carefully pipette supernatant off and discard.
?. [TRIzol Elution of RNA]
Airdry the RNA pellet.
Air dry the RNA pellet by leaving tubes open in fume hood to prevent contamination. This will take ~3-10 min depending on how much ethanol is left in the tube. Do not to let the RNA pellet dry completely as this will greatly decrease its solubility.
?. [Off-Bead Reverse Transcription]
Resuspend RNA pellet in  RT resuspension mix:  AB1ReagentVolume2DEPC-treated ddH2O8.7 µL3Primer RP1 (10 µM)4 µL4dNTP mix (12.5 mM each)0.8 µL
13.5 µl
AB1ReagentVolume2DEPC-treated ddH2O8.7 µL3Primer RP1 (10 µM)4 µL4dNTP mix (12.5 mM each)0.8 µL
Reverse transcription can also be performed on-bead, but we find that this significantly reduces library yield while increasing adapter dimer. For this reason, it is not recommended except in cases where material is abundant (107 cells) and speed is paramount. To do this, follow steps 49 and 50, then skip to step 65, but resuspend the beads instead of the RNA pellet in RT resuspension mix. After RT, elute cDNA by heating the bead mixture to 95°C, quickly place tubes on a magnet stand, and remove and save supernatant. Resuspend beads in 20 µL ddH2O and repeat the process for a final volume of 40 µL. Proceed with PreCR but use 20 µL less ddH2O (13.5 µL) in the PreCR mix and use the entire 40 µL eluate instead of the 20 µL RT mix.
?. [Off-Bead Reverse Transcription]
1.Denature at  for  and snap cool .
65 °C
on ice
?. [Off-Bead Reverse Transcription]
1.Prepare RT master mix:  AB1ReagentVolume25X RT Buffer4 µL3100 mM DTT1 µL4SUPERase-In RNase Inhibitor0.5 µL5Maxima H Minus RT enzyme1 µL
AB1ReagentVolume25X RT Buffer4 µL3100 mM DTT1 µL4SUPERase-In RNase Inhibitor0.5 µL5Maxima H Minus RT enzyme1 µL
?. [Off-Bead Reverse Transcription]
Add  to each sample (Vf = ).
6.5 µl
20 µl
?. [Off-Bead Reverse Transcription]
Cycle as follows: a.  for b.  for c.  for d. hold at .
50 °C
65 °C
85 °C
4 °C
?. [Off-Bead Reverse Transcription]
Immediately proceed to PreCR, test amplification, or full-scale amplification. Samples can be stored overnight at  (see Notes 38–39).
-20 °C
PreCR is optional if full scale amplification will be performed within 2 days. Longer storage of single-stranded cDNA libraries can lead to loss of library material. If you are skipping PreCR, simply store the 20 µL RT reaction at -20°C overnight and perform test amplification the next day.
Because this protocol uses molecular barcodes (UMIs) which facilitate robust computational PCR deduplication, it is less important to precisely determine the optimal cycle number. We recommend performing test amplification the first time you perform this protocol with a given amount of material from a given cell line to determine the optimal cycle number. For future experiments where the material and cell number are constant, test amplification can be skipped. Adjust the volume of the full-scale PCR to 100 µL total volume (accounting for the fact that the written protocol assumes loss due to test amplification). Test amplification can be performed either by PCR of a dilution curve and PAGE analysis or qPCR.
?. [PreCR]
Because this protocol uses molecular barcodes (UMIs) which facilitate robust computational PCR deduplication, it is less important to precisely determine the optimal cycle number. We recommend performing test amplification the first time you perform this protocol with a given amount of material from a given cell line to determine the optimal cycle number. For future experiments where the material and cell number are constant, test amplification can be skipped. Adjust the volume of the full-scale PCR to 100 µL total volume (accounting for the fact that the written protocol assumes loss due to test amplification). Test amplification can be performed either by PCR of a dilution curve and PAGE analysis or qPCR.
?. [PreCR]
Add  RPI-n indexed primer () to each sample. Use different barcodes for samples that will be pooled and sequenced together.
2.5 µl
?. [PreCR]
Prepare the PreCR master mix:  AB1ReagentVolume2ddH2O33.5 µL35X Q5 Buffer20 µL45X Q5 Enhancer20 µL5Primer RP1 (10 µM)1 µL6dNTP mix (12.5 mM each)2 µL7Q5 Polymerase1 µL
AB1ReagentVolume2ddH2O33.5 µL35X Q5 Buffer20 µL45X Q5 Enhancer20 µL5Primer RP1 (10 µM)1 µL6dNTP mix (12.5 mM each)2 µL7Q5 Polymerase1 µL
?. [PreCR]
Add  of the PreCR mix to each sample for final volume .
77.5 µl
100 µl
Do not attempt to scale down the PreCR or full-scale amplification steps to save PCR reagents. If RT reaction mixture exceeds 20% of the PCR reaction volume, significant inhibition of PCR will occur and lead to dramatically lower final library yield.
?. [PreCR]
Amplify libraries for 5 cycles on thermal cycler using the following settings:a.  for b.  for c.  for d.  for e. Go to b. 4 more timesf.  for g. Hold at
95 °C
95 °C
56 °C
72 °C
72 °C
4 °C
?. [PreCR]
Store samples at  or proceed to test amplification.
-20 °C
?. [Test Amplification (Gel)]
Make the first dilution using one of the following options:
Because this protocol uses molecular barcodes (UMIs) which facilitate robust computational PCR deduplication, it is less important to precisely determine the optimal cycle number. We recommend performing test amplification the first time you perform this protocol with a given amount of material from a given cell line to determine the optimal cycle number. For future experiments where the material and cell number are constant, test amplification can be skipped. Adjust the volume of the full-scale PCR to 100 µL total volume (accounting for the fact that the written protocol assumes loss due to test amplification). Test amplification can be performed either by PCR of a dilution curve and PAGE analysis or qPCR
Taking 7.7 µL of the 100 µL PreCR reaction leaves 92.3 µL for full-scale amplification. 25% of material in the first dilution is lost to make the next serial 4-fold dilution (2 of 8 µL). Because (7.7 * 0.75) / 92.3 ≈ 1/16, this first dilution is equivalent to the number of test amplification cycles less 4. If starting from the RT reaction, the volume has been adjusted for 5-fold lower starting volume.
?. [Test Amplification (Gel)]
If PreCR was performed, add  of the  PreCR reaction to  ddH2O for a final volume of .
7.7 µl
100 µl
0.3 µl
8 µl
?. [Test Amplification (Gel)]
If PreCR was skipped, add  of the  RT reaction to  ddH2O for a final volume of .
1.54 µl
20 µl
6.46 µl
8 µl
?. [Test Amplification (Gel)]
Make 4-fold serial dilutions by adding  of each dilution to  ddH2O for the next dilution.
2 µl
6 µl
?. [Test Amplification (Gel)]
Remove and discard  from the final dilution (all dilutions should now be ).
2 µl
6 µl
?. [Test Amplification (Gel)]
Choose a target number of total cycles for test amplification using the table below (see Note 42). The first dilution simulates full-scale amplification at the total number of cycles (PreCR cycles + Test Amp cycles) minus 4. Subtract 2 cycles sequentially for the following dilutions.  ABCDEFGHI1Dilution12345678219 Total Cycles15131197531321 Total Cycles171513119753423 Total Cycles1917151311975
ABCDEFGHI1Dilution12345678219 Total Cycles15131197531321 Total Cycles171513119753423 Total Cycles1917151311975
Additional cycles can vary by cell type. For HeLa, we typically perform 14 additional cycles (19 total cycles), which simulates 15 full-scale amplification cycles. For low input libraries (50k-250k mammalian cells), we typically perform 20 additional cycles (23 cycles total), which simulates 21 full-scale amplification cycles.
?. [Test Amplification (Gel)]
Make test PCR mix:  AB1ReagentVolume2ddH2O4.4 µL35X Q5 Buffer4 µL45X Q5 Enhancer4 µL5Primer RP1 (10 µM)0.5 µL6Primer RPI-n (10 µM)0.5 µL7dNTP mix (12.5 mM each)0.4 µL8Q5 Polymerase0.2 µL
AB1ReagentVolume2ddH2O4.4 µL35X Q5 Buffer4 µL45X Q5 Enhancer4 µL5Primer RP1 (10 µM)0.5 µL6Primer RPI-n (10 µM)0.5 µL7dNTP mix (12.5 mM each)0.4 µL8Q5 Polymerase0.2 µL
?. [Test Amplification (Gel)]
Add  PCR mix to the  diluted test samples (Vf = ).
14 µl
6 µl
20 µl
?. [Test Amplification (Gel)]
Amplify reactions for the desired amount of cycles using following settings:a.  for b.  for c.  for d.  for e. Go to step 2 for the desired number of cyclesf. for g. Hold at
CRITICAL Remember to account for PreCR. Subtract 5 cycles from your total target test amplification cycles.
95 °C
95 °C
65 °C
72 °C
72 °C
4 °C
If PreCR was skipped, use an annealing temperature of 56°C for the first 5 cycles of test amplification and the full-scale amplification.
?. [Test Amplification (Gel)]
Mix with gel loading dye to 1X and run  on a  Agarose gel or run  on a native  polyacrylamide gel and stain with SYBR Gold.
10 µl
2 µl
?. [Test Amplification (Gel)]
Use the test amplification gel to determine the appropriate number of cycles for full-scale amplification.
Desired amplification characteristics include a sufficient amount of product (smear starting ~150 bp), no evidence of overamplification, and ~50% primer exhaustion. The adaptor dimer product is 132 bp, and the smear will start 15–20 bp above this band. RNA degradation will lead to shorter library products. See:
?. [3.14	Test Amplification (qPCR)]
Add  of the  RT reaction to  ddH2O (Vf = ).
Because this protocol uses molecular barcodes (UMIs) which facilitate robust computational PCR deduplication, it is less important to precisely determine the optimal cycle number. We recommend performing test amplification the first time you perform this protocol with a given amount of material from a given cell line to determine the optimal cycle number. For future experiments where the material and cell number are constant, test amplification can be skipped. Adjust the volume of the full-scale PCR to 100 µL total volume (accounting for the fact that the written protocol assumes loss due to test amplification). Test amplification can be performed either by PCR of a dilution curve and PAGE analysis or qPCR
1.54 µl
20 µl
0.46 µl
2 µl
?. [3.14	Test Amplification (qPCR)]
Make the qPCR master mix:  AB1ReagentVolume2Primer RP1 (10 µM)0.25 µL3Primer RPI-n (10 µM)0.25 µL42X SsoAdvanced Universal SYBR Green Supermix5 µL5ddH2O2.5 µL
AB1ReagentVolume2Primer RP1 (10 µM)0.25 µL3Primer RPI-n (10 µM)0.25 µL42X SsoAdvanced Universal SYBR Green Supermix5 µL5ddH2O2.5 µL
?. [3.14	Test Amplification (qPCR)]
Add  of the qPCR master mix to  diluted RT reaction (Vf = ).
8 µl
2 µl
10 µl
?. [3.14	Test Amplification (qPCR)]
Quickly spin plate to collect liquid.
?. [3.14	Test Amplification (qPCR)]
Amplify in a real-time PCR system using the following conditions:
?. [3.14	Test Amplification (qPCR)]
Amplificationa.  for b.  for c.  for d. Go to step 2 for 39 additional cycles
98 °C
98 °C
60 °C
?. [3.14	Test Amplification (qPCR)]
Melt Curvea. for b.  for c.  for d.  for
95 °C
60 °C
96 °C
60 °C
?. [3.14	Test Amplification (qPCR)]
Calculate the number of full-scale amplification cycles as the cycle number where Rn reaches 0.25 × Rnmax.
?. [Full-Scale Amplification]
If PreCR and Test Amplification were skipped:
?. [Full-Scale Amplification]
Add  of an RPI-n indexed primer () to each  RT reaction. Use different barcodes for samples that will be pooled and sequenced on a single lane.
2.5 µl
20 µl
?. [Full-Scale Amplification]
Prepare the PCR master mix:  AB1ReagentVolume2ddH2O33.5 µL35X Q5 Buffer20 µL45X Q5 Enhancer20 µL5Primer RP1 (10 µM)1 µL6dNTP mix (12.5 mM each)2 µL7Q5 Polymerase1 µL
AB1ReagentVolume2ddH2O33.5 µL35X Q5 Buffer20 µL45X Q5 Enhancer20 µL5Primer RP1 (10 µM)1 µL6dNTP mix (12.5 mM each)2 µL7Q5 Polymerase1 µL
Do not attempt to scale down the PreCR or full-scale amplification steps to save PCR reagents. If RT reaction mixture exceeds 20% of the PCR reaction volume, significant inhibition of PCR will occur and lead to dramatically lower final library yield.
?. [Full-Scale Amplification]
Add  PCR master mix to each sample for final volume .
77.5 µl
100 µl
?. [Full-Scale Amplification]
Run the desired number of cycles:a.  for b.  for c.  for d.  for e. Go to step (b) for 4 more cycles.f.  for g.  for h.  for i. Go to step (f) for the desired number of cyclesj. Hold at
95 °C
95 °C
56 °C
72 °C
95 °C
65 °C
72 °C
4 °C
?. [Full-Scale Amplification]
If PreCR was skipped but Test Amplification was performed:
?. [Full-Scale Amplification]
Add  of an RPI-n indexed primer () to the remaining  RT reaction. Use different barcodes for samples that will be pooled and sequenced on a single lane.
2.5 µl
18.5 µl
?. [Full-Scale Amplification]
Prepare the PCR master mix:  AB1ReagentVolume2ddH2O35 µL35X Q5 Buffer20 µL45X Q5 Enhancer20 µL5Primer RP1 (10 µM)1 µL6dNTP mix (12.5 mM each)2 µL7Q5 Polymerase1 µL
AB1ReagentVolume2ddH2O35 µL35X Q5 Buffer20 µL45X Q5 Enhancer20 µL5Primer RP1 (10 µM)1 µL6dNTP mix (12.5 mM each)2 µL7Q5 Polymerase1 µL
Do not attempt to scale down the PreCR or full-scale amplification steps to save PCR reagents. If RT reaction mixture exceeds 20% of the PCR reaction volume, significant inhibition of PCR will occur and lead to dramatically lower final library yield.
?. [Full-Scale Amplification]
Add  PCR master mix to each sample for final volume .
79 µl
100 µl
?. [Full-Scale Amplification]
Run the desired number of cycles:a.  for b.  for c.  for d.  for e. Go to step (b) for 4 more cycles.f.  for g.  for h.  for i. Go to step (f) for the desired number of cyclesj. Hold at
95 °C
95 °C
56 °C
72 °C
95 °C
65 °C
72 °C
4 °C
?. [Full-Scale Amplification]
If PreCR and Test Amplification were performed:
?. [Full-Scale Amplification]
Prepare the following spike-in PCR mix:  AB1ReagentVolume2ddH2O3.7 µL35X Q5 Buffer1.5 µL45X Q5 Enhancer1.5 µL5dNTP mix (12.5 mM)0.5 µL6Q5 Polymerase0.5 µL
AB1ReagentVolume2ddH2O3.7 µL35X Q5 Buffer1.5 µL45X Q5 Enhancer1.5 µL5dNTP mix (12.5 mM)0.5 µL6Q5 Polymerase0.5 µL
?. [Full-Scale Amplification]
Add  PCR spike-in mix to each sample for final volume .
7.7 µl
100 µl
?. [Full-Scale Amplification]
Run the desired number of cycles. a.  for b.  for c.  for d.  for e. Go to step (b) for the desired number of cyclesf. Hold at
95 °C
95 °C
65 °C
72 °C
4 °C
Remember to account for PreCR. Subtract 5 cycles from your total target full-scale amplification cycles.
?. [Full-Scale Amplification]
Allow PCR reactions to reach room temperature.
?. [Full-Scale Amplification]
Add  SPRI beads (see Note 45) at room temperature and immediately mix by pipetting > 15X.
180 µl
?. [Full-Scale Amplification]
Incubate at  for .
4 Room temperature
?. [Full-Scale Amplification]
Place on a magnet stand and remove the supernatant.
?. [Full-Scale Amplification]
Wash the beads twice with ethanol without resuspending.
Do not disturb the beads or library recovery will be greatly reduced.
?. [Full-Scale Amplification]
Airdry the beads for . Do not over dry the beads.
?. [Full-Scale Amplification]
Resuspend beads in  Tris-Cl,  (no EDTA).
22 µl
?. [Full-Scale Amplification]
Incubate at room temperature for .
?. [Full-Scale Amplification]
Place the beads on a magnet stand and transfer  to a new tube.
20 µl
?. [Full-Scale Amplification]
Quantify the library using the Qubit dsDNA-HS assay and run on a Bioanalyzer.
?. [PAGE purification]
Due to advances in streptavidin bead technology and titration of adapters presented in this protocol, PAGE purification is rarely necessary. We prefer to sequence libraries that are 0%–25% adapter dimer rather than risk size bias associated with gel purification. Only perform PAGE purification if absolutely necessary. If needed, multiple libraries can be pooled by molarity as determined by bioanalyzer and extracted from the same gel lane to minimize size bias.
?. [PAGE purification]
Add Orange G loading dye to 1X to the entire library volume.
?. [PAGE purification]
Run the samples on a native polyacrylamide gel.
?. [PAGE purification]
Stain with SYBR Gold.
?. [PAGE purification]
Cut a gel slice from immediately above the adapter dimer to ~650 bp.
Desired amplification characteristics include a sufficient amount of product (smear starting ~150 bp), no evidence of overamplification, and ~50% primer exhaustion. The adaptor dimer product is 132 bp, and the smear will start 15–20 bp above this band. RNA degradation will lead to shorter library products. See:
?. [PAGE purification]
Place the gel slice in a 0.5 mL microfuge tube.
?. [PAGE purification]
Make a hole in the bottom of the tube with an 18G needle.
?. [PAGE purification]
Nest the 0.5 mL tube in a 1.5 mL tube and spin at 5000 x g for .
?. [PAGE purification]
If gel remains in the 0.5 mL tube, repeat step 7 and pool shredded gel fractions by suspending each in  soaking buffer using a wide-bore P1000 tip.
250 µl
?. [PAGE purification]
Soak the gel pieces in  soaking buffer (TE +  NaCl +  Tween-20) overnight with agitation at .
0.5 ml
37 °C
?. [PAGE purification]
Spin the tube at 5000 x g for 1 min.
?. [PAGE purification]
Pipette as much of the soaking buffer as possible without transferring gel pieces into a new tube.
?. [PAGE purification]
Add an additional  soaking buffer and incubate  at  with agitation.
0.5 ml
37 °C
?. [PAGE purification]
Spin the tube at 5000 x g for .
?. [PAGE purification]
Pipette as much of the soaking buffer as possible without transferring gel pieces into the tube with the previous eluate.
?. [PAGE purification]
Pass the remaining gel solution through a Costar Spin-X column using a cut P1000 tip and pool with the previous eluate (Vf = )
1 ml
?. [PAGE purification]
Reduce the volume by half (Vf = ) using vacuum dryer at .
0.5 ml
37 °C
?. [PAGE purification]
Add  GlycoBlue.
1 µl
?. [PAGE purification]
Add 2.5X volume ()  ethanol and vortex.
1.25 ml
?. [PAGE purification]
Centrifuge at >20,000 x g for  at .
4 °C
A blue pellet should be visible at the bottom of tube. The pellet can be difficult to see but should be visible. It may appear spread out. If a pellet is not visible, vortex well and repeat spin.
?. [PAGE purification]
Carefully pipette off the supernatant and discard.
When removing the supernatant before the 70% ethanol wash be careful not to disturb the pellet. Approximately 30–50μL of ethanol can be left in the tube to avoid disturbing the pellet prior to adding the 70% ethanol wash. This procedure can also be used after the 70% ethanol wash, but then remove the final 30-50 µL using a P200 tip after a quick spin in a picofuge.
?. [PAGE purification]
Add  of  ethanol.
750 µl
?. [PAGE purification]
Mix by gentle inversion and quickly spin down.
?. [PAGE purification]
Carefully pipette off the supernatant and discard.
?. [PAGE purification]
Air-dry the RNA pellet.
Air dry the RNA pellet by leaving tubes open in fume hood to prevent contamination. This will take ~3-10 min depending on how much ethanol is left in the tube. Do not to let the RNA pellet dry completely as this will greatly decrease its solubility.
?. [PAGE purification]
Resuspend the pellet in the desired volume of  Tris-Cl, , no EDTA
?. [Computational Analysis]
A pipeline for alignment of PRO-seq data can be found here: https://github.com/JAJ256/PROseq_alignment.sh