Probe amplification using T7

Updated 02/08/22

Linear PCR: pre-amplification of the entire library

Twist recommends ~10 ng template. That’s 0.2 ng/µL as the final template concentration for a 50 µL reaction.

2X linear PCR:

Reagent	Initial	Final	1X reaction (50 μl)	2X (μl)
Roche Diagnostics KAPA HiFi HotStart ReadyMix PCR Kit (6.25 mL), 250 x 50 uL reactions	2x	1x	25	50
Forward	10 µM	0.4 µM	2	4
Reverse (with T7 promoter)	10 µM	0.4 µM	2	4
Raw library	2 ng/µL	0.2 ng/µL	5	10
Ultrapure Water			16	32

1. Linear PCR program –95C-5min, (95C-30s, 58C-30s, 72C-20s)X20, 72C-5min
2. Purify with Zymo-5 kit, resuspend in ~30 ul of water
3. Expected yield: ≥ 50 ng/ul

Purify with Zymo 5 (centrifugation) (or Zymo 100 for the 96X reaction). Concentrate into 1 tube by repeating the DNA elution step and then re-using the elute for eluting the following sample. Elute with 30μl UPW.

50X Linear PCR:

Reagent	Initial	Final	1X (50 uL)	50X (2.5 uL)
Roche Diagnostics KAPA HiFi HotStart ReadyMix PCR Kit (6.25 mL), 250 x 50 uL reactions	2x	1x	25	1,250
Forward	10 µM	0.4 µM	2	100
Reverse (T7)	10 µM	0.4 µM	2	100
Linear-amplified library	2 ng/µL	0.2 ng/µL	5	250
Water			16	800

1. Combine the wells into a single 50 mL tube.
2. Add 2X DNA binding buffer (5 mL for a 50X reaction, Zymo kit).
3. Add 4X 100% Ethanol (10 mL for a 50X reaction).
4. Transfer to vacuum column and vacuum.
5. Wash twice with 2 mL of DNA washing buffer (Zymo).
6. Transfer the column into a 1.5 mL Eppendorf tube and spin at top speed for 30 seconds to remove excess liquid.
7. Elute in 150 μl UPW.
8. Spec in NanoDrop with 1.5 μl.

T7 Reaction (Adapted from Spatially resolved, highly multiplexed RNA profiling in single cells, K. H. Chen, A. N. Boettiger, J.R. Moffitt, S. Wang, and X. Zhuang. Science, 348 (6233) ,2015. And modified by Son. C. Nguyen

• Recommended to have at least 1 ug of DNA per 10.5 ul. If DNA concentration is lower, it is recommended to SpeedVac, and then resuspend to at least 1 ug/10.5 ul. I am using 120 ng/ul. Also recommended to run a 4x reaction.
• With PCR product containing a T7 promoter

Reagent	Initial	Final	1x reaction (μl)	4x reactions (μl)
DNA (120 ng/μl)			10.5	42
ATP (provided by kit)			3	12
CTP (provided by kit)			3	12
GTP (provided by kit)			3	12
UTP (provided by kit)			3	12
T7 buffer			3	12
RNaseOUT			1.5	6
T7 Pol Mix			3	12
Total			30	120

• Incubate at 37^oC overnight at the PCR machine. First close cap and then tight. Press instant incubate and follow instructions. If using a 1.5 ml tube then use a heated-lead dry bath.
• Following overnight T7 reaction:

RT PCR:

Reagent	Initial	Final	1x (μl)	4x reactions (μl)
T7 sample			30	120
Water			9.4	37.6
Forward primer	200 uM	1500 pmol	7.5	30
dNTP	25 mM each NTP	3.2 mM each NTP	9.6	38.4
RT buffer	5X	1X	15	60
RNaseOUT			1.5	6
Maxima RT -H			2	8
Total			75	300

• RT program on PCR machine: (Run -> GUY -> RT1 -> thick -> 50 μl (for 4X reaction)). Details of the PCR program:
  1. Incubate for 2 hours at 50 ^oC
  2. Inactivate RT at 85 C for 5 min.

1. Digestion – For unlabeled and labeled Oligopaints with the following dyes – Cy3:
   1. Mix 0.5 M EDTA and 1 M NaOH 1:1, 1X reaction volume (75 μl, or 300 μl for 4X reaction). Final NaOH concentration: 0.25 M. Total reaction volume: 150 μl.
   2. Heat at 95c for 10 minutes. If using a 1.5 ml tube then use a water bath and place cap-sealer on the cap. Once the reaction is done, put on ice and don’t remove the cap until the tube gets cooler.
2. Probe Cleanup:
   1. Combine the 6 tubes from the digestion reaction into a single 15 mL tube.
   2. Add 2X oligo binding buffer (300 μl or 1.2 ml for 4X reaction, Zymo kit).
   3. Add 4X 100% Ethanol (600 μl or 2.4 ml for 4X reaction).
   4. Transfer to vacuum column and vacuum.
   5. Wash twice with 2 ml of DNA washing buffer (Zymo).
   6. Transfer column into 1.5 Eppendorf and spin at top speed for 30 seconds to remove excess liquid.
   7. Elute in 200 μl UPW and spec.
   8. Calculate yield – In 1 reaction there is 1500 pmole primer. Translate the nanodrop read to pmoles and divide total probes in pmoles with primer in pmoles.
3. Speedvac and resuspend to 200 pmol/μl.

Gel electrophoresis:
We now use precast agarose gels. It takes a few minutes to set and 10 minutes to run.

For standard agarose gels, follow these guidelines:

1. Run 2% gel: Prepare 60 ml TAE, 1.2 gr Agar. Microwave until the solution is clear.
2. Add 6μl of 10,000X SyberSafe.
3. Spray gel tray walls with ethanol to allow the easier placing of gel tray in the container. Don’t forget comb. Turn gel tray.
4. Mix 4 μl  DDW, 1μl 1kb DNA Ladder (Invitrogen; 1 μg/lane), 1 μl 6X loading dye.
5. Mix >200 ng of sample with 6X loading dye.
6. Mix 4 μl  DDW, 1μl 100bp DNA Ladder (NEB; 0.5 μg/lane), 1 μl 6X loading dye.
7. Remove comb.
8. Add 1X TAE up to the fill line.
9. Load samples.
10. Run with 105 mV for an hour.

1. Digestion for labeled Oligopaints:
   1. RNase A, H treatment: 37 oC for 1 hr
2. Ethanol precipitate – assuming reaction volume of 75 μl, overnight at -20^oC, or at least 1 hr at -80^oC

• 4 M Ammonium acetate (1:10 of reaction volume) = 7.5 μl
• 100% ethanol (2.5:1) = 187.5 μl
• Glycogen (1:50) = 1.5 μl

• Ethanol precipitate step B – Spin at 4^oc (centrifuge in the cold room) at maximum speed for 30 minutes. Insert PCR tubes into Eppendorf tubes and balance. If required to change the rotor, then first press short spin, then the centrifuge will give a warning and adjust to the new rotor. Then, adjust your parameters (30 min, max speed).
• Ethanol precipitate step C – take an aliquot of 70% Ethanol from -20^o Discard suspension and add 500 ml 70% ethanol (wash), point the pipette tip to the walls to avoid resuspending the pellet. Discard again. Place tubes upside down with their cap open on a Kim wipe to evaporate ethanol residues. Then gently vacuum the walls of the tubes. Stay away from the pellet. Elucidate in 50 μl of water.
• Read concentration on Nanodrop. Concentration shouldn’t be higher then probe concentration. Nanodrop read is skewed due to Ethanol precipitation.