hyRAD RNA probes preparation and capture

Choose a few fresh samples coming from different populations (if possible, to capture the diversity of the targeted species). The DNA samples should be diluted to the same concentrations, ideally at 20-50 ng/μl. If you need to sequence the probes library, you should keep the samples separated for the next steps, until they are indexed in a PCR.

Prepare master mix 1:

66

10

2

2

Mix 20 μl of DNA with 80 μl of master mix A1 (total volume = 100 μl).

Incubate 3 hours at 37°C, hold at 4°C.

Perform AMPure cleanup with the beads:sample ratio 2:1 according to the manufacturer’s instructions. Resuspend in 20 μl of 10 mM Tris.

Prepare RAD-P1 adapters. You need to anneal:

1. RAD-P1.1_T7-PstI with RAD-P1.1_T7-Pst2 to obtain RAD-T7-P1 adapter (10 μM)
2. RAD_P2.1_MseI-bio with RAD_P2.2_MseI to obtain RAD-P2 adapter (10 μM)

A	B
reagent	volume [μl]
first oligonucleotide (100 μM)	10.0
second oligonucleotide (100 μM stock)	10.0
RNase-free water	70.0
Annealing buffer (10x)	10.0

Heat the mix for 1 min at 95°C, cool down to 10°C with ramp 0.1°C/s.

The annealed adapters can be stored long-term at -20°C.

Prepare master mix 2:

0.2

2.8

2.0

Mix 19 μl of digested DNA with 2 μl of RAD-T7-P1 adapter (10 μM) and 2 μl of RAD-P2 adapter (10 μM). Briefly vortex and spin.

Add 5 μl of master mix 2 to the digested DNA with adapters (total volume = 28 μl). Briefly vortex and spin.

Incubate 3 hours at 16°C, hold at 4°C.

Perform AMPure cleanup with the beads:sample ratio 1.5:1 according to the manufacturer’s instructions. Resuspend in 60 μl of 10 mM Tris.

Perform Pippin Prep size selection using 30 ul of the sample according to the instructions, keeping another 30 μl in case the size selection fails. You obtain 40 μl of the size selected sample. 

Measure concentration and check the profile of the sample using Fragment Analyzer/Tapestation/Bioanalyzer.

Mix well Dynabeads C1, make sure that all the beads are resuspended.

Dispense 30 μl of beads in a PCR tube (for each capture).

Wash 1:

1. put on the magnetic rack, wait until the beads separate,
2. remove and discard supernatant,
3. take the tube off the magnetic rack,
4. resuspend in 1 ml of 2x Bind and Wash buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 2 M NaCl).

Wash 2:

1. put on the magnetic rack, wait until the beads separate,
2. remove and discard supernatant,
3. take the tube off the magnetic rack,
4. resuspend in 1 ml of 2x Bind and Wash buffer.

Wash 3:

1. put on the magnetic rack, wait until the beads separate,
2. remove and discard supernatant,
3. take the tube off the magnetic rack,
4. resuspend in 40 μl of 2x Bind and Wash buffer.

Combine 40 μl of the size-selected ligation product with 40 μl of the beads in 2x Bind and Wash buffer.

Incubate 15 min at room temperature with gentle agitation. In the meantime, prepare the 1x wash buffer (see next step).

For each sample, prepare 3 ml of 1x Bind and Wash buffer by mixing 1.5 ml of 2x Bind and Wash buffer and 1.5 ml of water.

Wash 1:

1. put on the magnetic rack, wait until the beads separate,
2. remove and discard supernatant,
3. take the tube off the magnetic rack,
4. resuspend in 1 ml of 1x Bind and Wash buffer.

Wash 2:

1. put on the magnetic rack, wait until the beads separate,
2. remove and discard supernatant,
3. take the tube off the magnetic rack,
4. resuspend in 1 ml of 1x Bind and Wash buffer.

Wash 3:

1. put on the magnetic rack, wait until the beads separate,
2. remove and discard supernatant,
3. take the tube off the magnetic rack,
4. resuspend in 1 ml of 1x Bind and Wash buffer.

Resuspend:

1. put on the magnetic rack, wait until the beads separate,
2. remove and discard supernatant,
3. take the tube off the magnetic rack,
4. resuspend in 30 μl of 10 mM Tris.

Prepare indexed primer solutions (see Before starting). Use different combination of indexed primers for each starting sample.

Prepare master mix 3 for 3 PCR reactions:

7.0

12.5

1.5

1.5

Mix 2.5 μl of the size selected ligation product with 22.5 μl of master mix 3 (total volume = 25 ul). Prepare 3 PCR reactions for each sample like this.

PCR program for KAPA HiFi HotStart ReadyMix:

3 min at 95°C

15 cycles* of:

• 20 s at 98°C
• 15 s at 60°C
• 30 s at 72°C 5 min at 72°C

hold at 4°C.

• optimize the number of PCR cycles. Overamplification leads to heteroduplex formation, visible on a gel as secondary peaks/bands. Product like this will sequence normally but is harder to quantify. Too many PCR cycles also increase chimera formation and PCR error rate.

Pool the triplicates of each sample and perform AMPure cleanup with the beads:sample ratio 1:1 according to the manufacturer’s instructions. Elute in 20 μl of 10 mM Tris.

Quantify the library concentration using Qubit. Concentration around 10 ng/μl should be enough.

If not enough of probes is obtained, the rest of size-selected library can be amplified or the PCR product can be re-amplified: dilute the purified PCR product 10x and use in PCR reactions.

Pool the PCR products obtained from each sample together in equimolar ratios.

Keep a small aliquot for verifying the restriction reaction (step 24) and aliqot for sequencing.

Prepare restriction digest reaction:

A	B
reagent	volume [μl]
water	3.35
CutSmart buffer (10x)	1.5
MseI (10,000 U/ml)	0.15
PCR product	10.0

Incubate for 3 hours at 37°C.

Check the restriction by running gel or Fragment Analyzer/Tapestation/Bioanalyzer on the sample before and after the restriction. There should be a visible peak of cut adapters and the main peak should shift accordingly.

In this step HiScribe T7 High Yield RNA Synthesis Kit is used. Usually 1 μl of the template yelds enough RNA. After the first reaction you can scale it up for the amount of probes needed.

Please make sure to read the HiScribeTM T7 Quick High Yield RNA Synthesis Kit Instruction Manual for instructions and troubleshooting.

Assemble the reaction:

A	B
reagent	volume [μl]
10X Reaction Buffer	1.5
ATP (100 mM)	1.5
GTP (100 mM)	1.5
CTP (100 mM)	1.5
UTP (100 mM)	1.0
Biotin-16-UTP (10 mM)	5.0
T7 RNA Polymerase Mix	1.5
DNA template	1.0
RNase-free water	5.5

Incubate at 37°C overnight (up to 16 h).

Add 1 μl TURBO DNase (4 U) and incubate at 37°C for 30 minutes.

Purify the RNA using RNEasy Mini kit (Qiagen) using standard protocol except adding 665 μl EtOH to the RNA + RTL mix before loading on the column and elute in 30 μl of RNase-free water.

Quantify using Qubit RNA kit. You can check the RNA size using Fragment analyzer/Tapestation or by TBE/Urea gel electrophoresis.

Dilute the RNA probes to around 100 ng/μl.

Add 1 μl of SUPERase-In for each 19 μl of the probes.

Store at −80°C if not used immediately.

Anneal the oligonucleotides used as the template for T7 polymerase reaction. You need to anneal:

1. blocking RNA template for P5 adapter with T7 promoter,
2. blocking RNA template for P7 adapter with T7 promoter.

A	B
reagent	volume [μl]
one of the two blocking oligos template (200 μM)	12.5
T7.prom oligo (200 μM)	12.5
RNase-free water	20.0
Annealing buffer (10x)	5.0

Heat the mix 1 min at 95°C, cool down to 10°C with ramp 0.1°C/s.

The annealed template can be stored long-term at -20°C.

Assemble the reaction for each annealed oligonucleotides pair:

A	B
reagent	volume [μl]
RNase-free water	10
10X Reaction Buffer	1.5
ATP (100 mM)	1.5
GTP (100 mM)	1.5
CTP (100 mM)	1.5
UTP (100 mM)	1.5
T7 RNA Polymerase Mix	1.5
Annealed template oligonucleotides	1

Incubate at 37°C overnight (up to 16 h).

Add 2 μl TURBO DNase (4 U) and incubate at 37°C for 30 minutes.

Purify the RNA using RNEasy Mini kit (Qiagen) using standard protocol except adding 665 μl EtOH to the RNA + RTL mix before loading on the column and elute in 30 μl of RNase-free water.

Quantify again using Qubit RNA kit. You can check the RNA size using Fragment analyzer/Tapestation or by TBE/Urea gel electrophoresis.

Adjust the concentration of each blocking RNA to 200 μM. This corresponds to the following concentrations:

1. BO.P5: 3129 ng/μl
2. BO.P5-multiplex: 4192 ng/μl
3. BO.P7-multiplex: 4366 ng/μl

Add 1 μl of SUPERase-In for each 19 μl of the final probes volume

Store at −80°C if not used immediately.

Mix the same amount of both blocking RNA oligonucleotides (BO.P5 and BO.P7-multiplex for single-indexed libraries or BO.P5-multiplex and BO.P7-multiplex for double-indexed libraries). The final concentration of each will be 100 μM.

Prepare the hybridization mix in the PCR tubes . Probes and blocking oligos are used in excess. So far, we tested the protocol using 500 ng of the genomic libraries and the same amount of the probes. For rare targets (aDNA for instance), you can increase library amount up to 2000 ng.

The below mixes are based on MyBaits protocol (http://www.mycroarray.com/pdf/MYbaits-manual-v3.pdf) and prepared in a bit larger quantities to account for evaporation and for easier handling.

Assemble the hybridization mix on ice:

A	B
reagent	volume [μl]
SSPE (20x)	9.0
EDTA (500 mM)	0.5
SDS (10%)	0.5
Denhardt’s solution (50x)	3.5
SUPERase-In (20 U/μL)*	1
hyRAD RNA probes (1000 ng)*	5.5

*keep on ice 

Total volume = 20.0 μl, of which 18.0 μl is used in the hybridization.

Prepare the blocking mix :

A	B
reagent	volume [μl]
Human Cot-1 DNA (1 mg/ml)	2.5
Salmon sperm DNA (1 mg/ml)	2.5
Mix of blocking RNA oligos	0.5

Total volume = 5.5 μl, of which 5.0 μl si used in the hybridization.

Put 7 μl of the prepared Illumina library in the PCR tube (500 ng), add 5 μl of the blocking mix . Mix by pippeting.

Program the termocycler for two steps (both steps with a heated lid):

1. 5 min at 95°C,
2. 16-24 h at hybridization temperature (see below)

Usual hybridization temperature is 65°C. For rare targets like aDNA, incubation can last for up to 40 hours at lower temprature of 60 or 55°C.

Incubate the library + blocking mix 5 min at 95°C (1st step).

When thermocycler temperature decrease to the hybridization temperature (2nd step), put the tubes with the hybridization mix in the thermocycler, incubate the library and hybridization mix for 5 more minutes at 65°C.

Still at the hybridization temperature, add 18 μl of the hybridization mix to the library+blocking mix and mix by pippeting. Total volume is now 30 μl. Do not take the mixture out from the PCR machine to vortex or centrifuge at this step.

Incubate at the hybridization temperature for 16-24 hours (up to 40 hours and lower temperature for rare targets like aDNA).

Mix well Dynabeads C1, make sure that all the beads are resuspended.

Dispense 30 μl of beads in a PCR tube (for each capture).

Wash 1:

1. put on the magnetic rack, wait until the beads separate,
2. remove and discard supernatant,
3. take the tube off the magnetic rack,
4. resuspend in 200 μl of TEN.

Wash 2:

1. put on the magnetic rack, wait until the beads separate,
2. remove and discard supernatant,
3. take the tube off the magnetic rack,
4. resuspend in 200 μl of TEN.

Wash 3:

1. put on the magnetic rack, wait until the beads separate,
2. remove and discard supernatant,
3. take the tube off the magnetic rack,
4. resuspend in 70 μl of TEN.

Store at room temperature until use.

Prepare the aliquots of the wash buffers and Dynabeads in TEN and prewarm them to the hybridization temperature in a thermocycler.

Keeping the hybridization reaction in a thermocycler at the hybridization temperature with the heated lid, add 70 ul of the Dynabeads in TEN to the 30 μl of the hybridization reaction and mix well by pipetting.

Incubate for 30 min at the hybridization temperature in a thermocycler.

0h 30m 0s

Wash 1:

1. put on the magnetic rack, wait until the beads separate,
2. remove supernatant,
3. resuspend beads in 180 μl of 1x SSC / 0.1% SDS prewarmed to the hybridization temperature,
4. mix well by vortexing, briefly centrifuge.

Incubate for 15 min at the hybridization temperature in a thermocycler.

0h 15m 0s

Wash 2:

1. put on the magnetic rack, wait until the beads separate,
2. remove supernatant,
3. resuspend beads in 180 μl of 0.1x SSC / 0.1% SDS prewarmed to the hybridization temperature,
4. mix well by vortexing, briefly centrifuge.

Incubate for 10 min at the hybridization temperature in a thermocycler.

0h 10m 0s

Wash 3:

1. put on the magnetic rack, wait until the beads separate,
2. remove supernatant,
3. resuspend beads in 180 μl of 0.1x SSC / 0.1% SDS prewarmed to the hybridization temperature,
4. mix well by vortexing, briefly centrifuge.

Incubate for 10 min at the hybridization temperature in a thermocycler.

0h 10m 0s

Wash 4:

1. put on the magnetic rack, wait until the beads separate,
2. remove supernatant,
3. resuspend beads in 180 μl of 0.1x SSC / 0.1% SDS prewarmed to the hybridization temperature,
4. mix well by vortexing, briefly centrifuge.

Incubate for 10 min at the hybridization temperature in a thermocycler.

0h 10m 0s

If you use Kapa HiFi Hot Start polymerase mix in the next steps you can keep the captured DNA on the beads and proceed to step 68.

Otherwise elute the DNA from the probes:

1. put on the magnetic rack, wait until the beads separate,
2. remove supernatant,
3. add 30 μl of 95°C 10 mM Tris,
4. mix well by pipetting.

Incubate for 10 min, 95°C in a thermocycler.

0h 5m 0s

Put on the magnet; remove and retain the supernatant .

This supernatant contains the hybridization-enriched products!

Discard the beads.

Prepare re-amplification master mix. The volumes are per one capture-enriched library:

A	B
reagent	volume [μl]
water	7.5
KAPA HiFi HotStart ReadyMix	25.0
PCR_F primer (5 μM)	5.0
PCR_R_post primer (5 μM)	5.0

Mix 42.5 μl of the master mix with 7.5 μl of the capture-enriched library.

PCR program for KAPA HiFi HotStart ReadyMix:

3 min at 95°C

15 cycles* of:

• 20 s at 98°C
• 15 s at 60°C
• 30 s at 72°C 5 min at 72°C

hold at 4°C.

• optimize the number of PCR cycles to obtain enough number of molecules for sequencing while using as few PCR cycles as possible.

Purify with AMPure using beads:sample ratio 1:1, elute in 30 μl of 10 mM Tris.

Verify the library profile and molarity using Fragment Analyzer/Tapestation/Bioanalyzer. Libraries can now be pooled in equimolar ratios.

Your libraries are now ready to be sent for sequencing, congratulations!