This protocol is originally from Mark Urich (SALK) and was modified by Stephane Lefebvre
(SFSU) to include a spike and optimised for T. pseudonana whole transcriptome library prep.
(stephane@rtc.sfsu.edu).
Please feel free to contact me if you have any questions, concerns, or comments about the
protocol. This is a work in progress and I will be updating it as I get feedback from others who
are referring to it. Thanks.
-Mark Urich
Required Kits
Poly(A)Purist Mag Kit
Quant-iT RNA Assay Kit
Quant-iT dsDNA HS Assay Kit
SOLiD Total RNA-Seq Kit
SOLiD RNA Barcoding Kit
RiboMinus Concentration Module
Agencourt AMPure XP
mRNA spike (Invitrogen AM1780M)
*Additional glycogen may be required.
*After RNA isolation determine the amount of total RNA in each sample using Nanodrop or
Quant-iT RNA BR Assay Kit. The Quant-iT RNA Assay Kit is only accurate from 5 - 100 ng/µL
while the BR kit is accurate from 20 - 1000 ng/µL. Store total RNA in elution buffer, TE, or
DEPC-Treated Water at -80˚ until ready for PolyA selection. Do not store long term in ethanol
and minimize freezing and thawing.
=====================================================================
Sampling
Filter about 100,000,000 cells on 0.2-1 µm pore size polycarbonate filters (45mm diameter).
Flash freeze in liquid N2 and store at -80oC.
Total RNA extraction
1.
2.
3.
4.
5.
6.
7.
8.
Re-suspend cells on the filter in 15mL tube containing 2 mL Trizol.
Transfer 2x ~1mL in two 1.5mL epp.
3. Incubate 5’@ RT
4. Add .2mL chloroform per 1mL Trizol, shake vigorously to emulsify
5. Incubate 2-3’@ RT
Spin 12,000g 15’ @ 4oC
Transfer clear top phase to fresh 1.5mL tube
Add 0.5mL isopropyl alcohol, mix by inversion 4-5 times, o/n @ -20oC.
9. Spin 12,000g 10’ @ 4oC
10. Discard supernatant and add 1mL 75%etOH, mix by inversion
11. Spin 12,000g 5’ @4oC
12. Discard supernatant
13. Air dry pellet 5-10’
14. Resuspend pellet in 50µL RNase free water (can store @-80oC here)
DNA digestion and clean up
1. Add 37.5 µL of RNase free water to the 50 µL from above
2. Add 10 µL RDD buffer and 2.5 µL DNase I
3. Incubate @ RT 10’
4. Add 350 µL Buffer RLT from RNeasy mini kit
5. Add 250 µL etOH 100%, mix by pipetting
6. Transfer supernatant to RNeasy mini spin column
7. Spin 8,000g 15’’, discard flow through
8. Add 500 µL RPE, spin 8,000g 15’’, discard flow through
9. Repeat step 8
10. Spin 2 ‘ at full speed
11. Place RNeasy column in 1.5 mL RNase free collection tube
12. Add 54 µL RNase free water
13. Incubate at RT 1-2’
14. Spin 8,000g, 1’
15. Quantify using Qubit, run Total RNA Nano chip
Total RNA profiles after Trizol extraction and RNA easy clean up
T. pseudonana Total RNA before
column purification
T. pseudonana Total RNA after
column purification
E. huxleyi Total RNA before column
purification
E. Huxleyi Total RNA after column
purification
Note: Poly(A)Purified mRNA will be ~1% of the total RNA
PolyA Selection Protocol
Ambion Poly(A)Purist Mag Kit
RNA Precipitation (optional)
Alcohol precipitate total RNA to remove residual salt (Perform this step using sticky tubes)
Add:
.1 volume 5 M Ammonium Acetate or 3 M Sodium Acetate
1µL Glycogen
2.5 volumes 100% ethanol
a. Precipitate at -80˚C overnight.
(Alternatively, to save time, tubes may be soaked in liquid nitrogen until the
precipitation solution becomes viscous.)
b. Recover RNA by centrifugation at max speed for 30 min at 4˚C.
c. Discard the supernatant.
d. Add 500 µL 70% ethanol, vortex the tube for a few seconds, and repellet by
centrifugation at max speed for 30 min at 4˚C.
e. Remove supernatant and let tubes dry open on the bench for 10 - 20 minutes.
RNA Prep (1st round poly(A) purification)
1.
2.
3.
Resuspend the RNA in Nuclease-free Water (1.5 µL per µg of total RNA). This
should give a final concentration of approximately 600 ng/µL.
(e.g. 30 µg total RNA = 45 µL Nuclease-free Water)
Add equal volume of 2x Binding Solution.
Preheat 310 µL of RNA Storage Solution (RNASS) for each sample at 70˚C
Prepare MagBeads
1.
2.
3.
4.
5.
Add .1 µL per µg total RNA of beads to supplied collection tubes.
(e.g. 30 µg total RNA = 3 µL MagBeads)
Add 50 µL Wash Solution #1 to beads.
Mix by flicking, pulse spin, then place on magnetic rack until solution is clear.
Remove supernatant by pipetting.
Repeat steps 3 through 5.
Bind to MagBeads
1.
2.
Add total RNA + Binding Buffer solution to MagBeads and mix gently by
pipetting.
Move to 70˚C incubator for 5 minutes.
3.
4.
Incubate at room temperature for 40 minutes.
(Constant agitation increases efficiency)
Move to magnetic rack for 2 minutes and remove supernatant.
Wash MagBeads
1.
2.
3.
4.
5.
6.
Add 50 µL of Wash Solution #1, mix by flicking, and pulse spin.
Move to magnetic rack until solution is clear and remove supernatant.
Repeat steps 1 and 2.
Add 50 µL of Wash Solution #2, mix by flicking, and pulse spin.
Move to magnetic rack until solution is clear and remove supernatant.
Repeat step 4.
Recover PolyA RNA
(It is best to perform this process with 4 or less samples at a time as they need to be done as
quickly as possible.)
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
Place new RNase-free collection tubes on magnetic rack. (for later use)
Move samples with Wash Solution #2 to magnetic rack and remove supernatant.
Remove samples from magnetic rack and place tubes in 70˚C incubator.
Add 150 µL 70˚C RNA Storage Solution.
Vortex for 2 full seconds, pulse spin if needed, and move to magnetic rack.
(It is normal for some bead clumps to not resuspend)
Remove supernatant and place in the new RNase-free tube on magnetic rack to
remove beads that may have carried over.
Add 150 µL 70˚C RNA Storage Solution to beads.
Repeat step 5.
Remove supernatant and place in same tube as the 1st RNA storage solution
elution.
Remove 300 µL RNA Storage Solution from tube on magnetic rack and place in
a new RNase-free tube.
RNA Prep (2nd round poly(A) purification)
1.
3.
Add equal volume of 2x Binding Solution (i.e.300µL)
Preheat 310 µL of RNA Storage Solution (RNASS) for each sample at 70˚C
Prepare MagBeads
1.
2.
3.
4.
5.
Add the same amount of beads as for 1st round to supplied collection tubes.
Add 50 µL Wash Solution #1 to beads.
Mix by flicking, pulse spin, then place on magnetic rack until solution is clear.
Remove supernatant by pipetting.
Repeat steps 3 through 5.
Bind to MagBeads
1.
2.
3.
4.
Add total RNA + Binding Buffer solution (600µL) to MagBeads and mix gently by
pipetting.
Move to 70˚C incubator for 5 minutes.
Incubate at room temperature for 40 minutes.
(Constant agitation increases efficiency)
Move to magnetic rack for 2 minutes and remove supernatant.
Wash MagBeads
1.
2.
3.
4.
5.
6.
Add 50 µL of Wash Solution #1, mix by flicking, and pulse spin.
Move to magnetic rack until solution is clear and remove supernatant.
Repeat steps 1 and 2.
Add 50 µL of Wash Solution #2, mix by flicking, and pulse spin.
Move to magnetic rack until solution is clear and remove supernatant.
Repeat step 4.
Recover PolyA RNA
(It is best to perform this process with 4 or less samples at a time as they need to be done as
quickly as possible.)
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
Place new RNase-free collection tubes on magnetic rack. (for later use)
Move samples with Wash Solution #2 to magnetic rack and remove supernatant.
Remove samples from magnetic rack and place tubes in 70˚C incubator.
Add 150 µL 70˚C RNA Storage Solution.
Vortex for 2 full seconds, pulse spin if needed, and move to magnetic rack.
(It is normal for some bead clumps to not resuspend)
Remove supernatant and place in the new RNase-free tube on magnetic rack to
remove beads that may have carried over.
Add 150 µL 70˚C RNA Storage Solution to beads.
Repeat step 5.
Remove supernatant and place in same tube as the 1st RNA storage solution
elution.
Remove 300 µL RNA Storage Solution( containing RNA poly(A) purified) from
tube on magnetic rack and place in a new RNase-free tube (Use sticky tubes).
Precipitate PolyA RNA
1.
2.
Add:
.1 volume 5 M Ammonium Acetate
1 µL Glycogen
2.5 volumes 100% ethanol
Store in -80˚C until library prep.
(30 µL)
(0.75 mL)
(Alternatively use liquid nitrogen precipitation if moving on to library prep immediately or standard o/n precipitation at -20˚C.)
Thalassiosira pseudonana mRNA chip after one and two rounds of Poly(A) selection
Eukaryote Total RNA Pico Chip
Thalassiosira pseudonana total RNA
Thalassiosira pseudonana after 1st round Poly(A) purification
Thalassiosira pseudonana after 2nd round Poly(A) purification
Eukaryote mRNA Pico Chip
Thalassiosira pseudonana after 2nd round Poly(A) purification
Remaining ribosomal RNA can be seen as peaks in the mRNAPico run.
Samples in the mRNAPico chip run are different from the samples in the RNAPico Chip run.
=====================================================================
SOLiD Total RNA-Seq Kit Protocol
Precipitation
1.
2.
3.
4.
5.
6.
Recover PolyA RNA by centrifugation at max speed for 30 min at 4˚C.
Discard the supernatant.
Add 500 µL 70% ethanol, vortex the tube for a few seconds, and repellet by
centrifugation at max speed for 30 min at 4˚C.
Remove supernatant and let tubes dry open on the bench for 10 - 20 minutes (or
spin vac to dry)
Resuspend in 10 µL DEPC-Treated Water
Check concentrations with Qubit fluorometer.
(This check is essential to add spike.)
Spiking & Fragmentation
1.
Place PolyA RNA tubes on ice
2.
In a new tube prepare:
1-7 µL of Poly(A) RNA (80ng)
1 µL Spike (100,000 transcripts)
1 µL 10x RNase III Buffer
1 µL RNase III
x µL DEPC-Treated Water to make up 10µL final
(Keep the RNase III and RNase III Buffer tubes on ice)
3.
Mix by flicking, spin pulse.
4.
Incubate at 37˚C for 10 minutes exactly.
5.
Prepare for RiboMinus Concentration Module.
(add 6 mL 100% ethanol to Wash Buffer)
Clean up fragmented PolyA RNA (Ribominus)
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
Immediately move samples to ice and add 90 µL of DEPC-Treated Water.
Add: 100 µL Binding Buffer
250 µL 100% ethanol
Load 450 µL to spin column.
Centrifuge at 12,000g for 1 minute. Discard flowthrough.
Add 500 µL Wash Buffer with ethanol to column.
Centrifuge at 12,000g for 1 minute. Discard flowthrough.
Centrifuge at max speed for 2 minutes.
Move column to new 1.5 mL RNase-free collection tube.
Add 12 µL DEPC-Treated Water to column.
Let column sit for 2 minutes. Centrifuge at max speed for 1 minute.
Check concentrations with Qubit fluorometer.
If concentrations are less than 17 ng / µL use a speed vac to concentrate.
Eukaryote mRNA Pico Chip
Thalassiosira pseudonana after fragmentation and purification
Ligation
(.5 kit protocol)
1.
2.
3.
2.
3.
4.
5.
Place new PCR tubes on ice
Prepare hybridization mix containing:
1 µL SOLiD Adapter Mix
1.5 µL Hybridization Solution
Add to PCR tubes:
2.5 µL hybridization mix
1.5 µL PolyA Fragmented RNA (25ng)
Mix thoroughly by pipetting.
Incubate at 65˚C for 10 minutes then 16˚C for 5 minutes.
Move PCR tubes back to ice and add in order:
5 µL 2x Ligation Buffer
1 µL Ligation Enzyme Mix
(Keep Ligation Enzyme Mix on ice)
Mix well and incubate at 16˚C for 16 hours.
Perform reverse transcription
(.5 kit protocol)
1.
2.
3.
On ice prepare RT master mix without the ArrayScript Reverse Transcriptase.
5.5 µL DEPC-Treated Water
2 µL 10x RT Buffer
1 µL dNTP mix
1 µL SOLiD RT Primer
Incubate the RT master mix with the ligated RNA sample:
a.
Add 9.5 μL of RT master mix to each 10-μL ligation reaction.
b.
Pipet up and down a few times to mix, then pulse spin briefly.
c.
Incubate in a thermal cycler with a heated lid at 70 °C for 5 minutes, then
snap-cool on ice.
Perform the reverse transcription reaction:
a.
b.
c.
Add .75 μL ArrayScript Reverse Transcriptase to each ligated RNA
sample. (Keep ArrayScript RT on ice)
Gently vortex to mix thoroughly, then pulse spin briefly.
Incubate in a thermal cycler with a heated lid at 42 °C for 30 minutes.
Purify the cDNA
(Agencourt AMPure XP)
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
Prepare new low-bind 1.5 mL tubes with 36 µL Ampure beads.
Add 20 µL cDNA to beads and mix by pipetting.
Incubate for 10 minutes at room temperature.
Move to magnetic rack until solution is clear and remove the supernatant.
Add 150 µL of freshly prepared 70% ethanol.
Wash beads by removing the tube from the magnetic rack, rotating 180˚ and
placing back on magnetic rack six times.
Remove the supernatant.
Repeat steps 5 and 7 two times.
Pulse spin and place back on the magnetic rack.
Remove any liquid left in the tubes.
Place in a hood until the beads are dry. (until cracks appear in the beads)
Add 10 µL DNA Extraction Buffer to beads and vortex.
Pulse spin and mix by pipetting.
Move back to magnetic rack and wait until the solution clears.
Remove supernatant and place in a new low-bind 1.5 mL tube on the magnetic
rack.
Amplify the cDNA
(.5x kit protocol)
1.
2.
3.
4.
5.
For each cDNA sample, prepare 98 μL PCR mix in a 0.2 mL PCR tube on ice:
76.8 µL
Nuclease-free Water
10 µL
10x PCR Buffer
8 µL
dNTP mix
2 µL
SOLiD 5’ PCR Primer
1.2 µL
AmpliTaq® DNA Polymerase (keep on ice)
Add 2 µL SOLiD 3’ PCR Primer from SOLiD RNA Barcoding Kit.
Add 5 µL of cDNA sample per reaction.
Run PCR reactions in a thermal cycler for 15 cycles:
Stage
Temp
Time
Hold
95˚C
5 min
Cycle 1
95˚C
30 sec
Cycle 2
62˚C
30 sec
Cycle 3
72˚C
30 sec
Hold
72˚C
7 min
After PCR store samples at 4˚C.
Purify the amplified DNA
(Ampure)
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
Prepare new low-bind 1.5 mL tubes with 180 µL Ampure beads.
Add 100 µL amplified DNA to beads and mix by pipetting.
Incubate for 10 minutes at room temperature.
Move to magnetic rack until solution is clear and remove the supernatant.
With tubes on the magnetic rack wash the beads with 300 µL 70% ethanol.
(you do not need to resuspend the beads)
Wash beads by removing the tube from the magnetic rack, rotating 180˚ and
placing back on magnetic rack six times.
Remove the supernatant.
Repeat steps 5 and 7 two times.
Pulse spin and place back on the magnetic rack.
Remove any liquid left in the tubes.
Place in a hood until the beads are dry. (until cracks appear in the beads)
Add 30 µL DNA Extraction Buffer to beads and vortex.
Pulse spin and mix by pipetting.
Move back to magnetic rack and wait until the solution clears.
Remove supernatant and place in a new low-bind 1.5 mL tube.
Add 54 µL Ampure beads to 30 µL purified DNA.
Repeat steps 3 through 15, using 10 µL DNA Extraction Buffer instead of 30 µL.
Check concentrations of DNA using Qubit fluorometer.
Library size distribution
Thalassiosira pseudonana library size distribution
Note: This library does not required gel size selection
Pool barcoded samples
(for multiplexing)
1.
2.
3.
4.
Determine the sample with lowest concentration.
Calculate the amount of DNA in 1 µL of the lowest sample.
Add that amount of DNA from each sample to a pool.
Make at least 10 µL of pooled libraries @ 10ng/µL for Andrew O'Shaughnessy.
Size select (optional)
1.
2.
3.
4.
5.
Run sample on a 2% low melting temperature gel.
Cut out 150 - 200bp region of sample.
Gel purify using Qiagen kit.
Resuspend in 12µL Buffer EB.
Check concentration of library using Qubit fluorometer.
Library dilution
1.
2.
3.
4.
Dilute the library to a concentration 1 ng/µL in 1x Low TE.
Check concentration of library using Qubit fluorometer.
Dilute the library to a concentration of 60 pg/µL in 1x Low TE.
Use 60 µL of the 60 pg/µL library for emulsion PCR.