Library preparation

Tissue sectioning

Before starting

Before sectioning, place the OCT-mounted fresh frozen tissue in a cryostat for 20 minutes at the selected cutting temperature (adjusted according to tissue). Place the capture areas at room temperature.

Before starting

Pre-cool 100% methanol at -20°C for subsequent fixation step.

Tip

Trim the excess OCT surrounding the tissue to prevent folding of OCT under or over the tissue.

Warning

Remove the capture area from the stage, as soon as the transfer occured, to avoid re-freezing of the tissue onto the stage.

1. Slice the tissue at the selected temperature at 10 μm thickness.
2. Place the capture area (room temperature) on the tissue section on the cutting stage. The tissue will melt onto the capture area.
3. Store the capture area with tissue-side up in the cryostat until fixation.

Fixation

Note

Transport samples on dry ice until placed in -20°C methanol.

1. Fix the tissue by placing the capture area with the tissue section in a tube containing 1 mL of 100% Methanol (pre-cooled to -20°C).
2. Incubate at -20°C for 30 min.

Hematoxilin and eosin (H&E) staining and imaging

Buffered Eosin (make fresh)

Reagent	Volume (μL)
Eosin Y	500
0.45M Tris-Acetate buffer pH 6.0	500

For incubations, add enough volume to cover the capture area completely. For all washes, wash by dipping in a beaker with Ultrapure water (500 mL).

1. Add isopropanol to the tissues on the capture area and incubate for 1 min, then remove the solution and dry the tissues.
2. Add hematoxylin, and incubate for 5 min.
3. Wash the capture area with Ultrapure water until the hematoxylin dye is completely removed (10-15x).
4. Add bluing buffer, and incubate for 2 min.
5. Wash the capture area with Ultrapure water, dipping 5 times.
6. Add buffered eosin onto tissue section for 1 min.
7. Wash the capture area with Ultrapure water, dipping 10-15x.
8. Let the capture area air-dry at room temperature for 10 min. Dry the tissues completely with no residual water.
9. Put the capture area face-down on a coverslip (#1.5, 24x50mm) and image on brightfield with the 20x objective.

After imaging, place the capture areas with the tissue section face up into a multi-well gasket, such as the 16-Well ProChamber Microarray System (Grace Bio-Labs, Cat#645508). We use 100 μL reaction volume throughout the protocol whilst using the gasket.

Permeabilization

We recommend performing a pilot experiment in which you compare different permeabilization conditions using a qPCR assay. We suggest comparing different pepsin incubation times (for example, 0, 15, 30, 45, and 60 min) at 37°C.
Follow the library preparation steps until qPCR for cycle number assessment. Earlier amplification corresponds to a higher concentration of starting material, ie. more efficient mRNA capture. If conditions amplify together, chose the shorter time or lower pepsin concentration for permeabilization of your sample.

1. Weigh and dissolve the pepsin to have a solution with 7 U/ul pepsin in 2xSSC pH 2.5.
2. Dilute 1:10 with 2xSSC pH 2.5 to get the final concentration of 0.7 U/μl.
3. Prewarm permeabilization mix at 37℃ several minutes before use.
4. Incubate at 37°C for X min (ex. 15 min - 30 min - 60 min) according to the tissue used.

Reverse transcription

Reverse transcription (RT) buffer

Reagent	Final concentration	Volume (μl)
SSIV 5X rt BUFFER	1x	20
RNase inhibitor (40U/ul)	1U/ul	2.5
Ultrapure water		77.5
Total		100

Reverse transcription mix

Reagent	Final concentration	Volume (μl)
SSIV 5X rt BUFFER	1x	20
0.1M DTT	5mM	5
BSA (20mg/ml)	0.187mg/ml	0.93
10mM dNTP mix	1mM	10
Superscript IV (200U/ul)	6.67 U/uL	3.33
Ribolock(40U/uL)	1U	2.5
Ultrapure water		58.24
Total		100

1. Remove the pepsin solution.
2. Wash the capture area carefully with 100 μl RT Buffer once.
3. Add 100 μL RT mix per capture area. Incubate overnight at 42°C.

Exo I digestion

Exonuclease I mix

Reagent	Final concentration	Volume (μl)
10 x Exo I buffer	1x	10
Exo I	1U/ul	5
Ultrapure water		85
Total		100

1. Remove the RT solution.
2. Add 100 μL Exonuclease I mix per capture area to eliminate DNA that did not hybridize with mRNA.
3. Incubate 45 min at 37℃.

Tissue removal

Tissue removal mix

Reagent	Final concentration	Volume (μL)
Tris-Cl pH 8.0	100 mM	10
2M NaCl	200 mM	10
20% SDS	2%	10
0.1M EDTA	5 mM	5
Proteinase K ( 800 mU/uL)	16 mU/uL	2
Nuclease -free water		63
Total		100

1. Remove the Exonuclease I mix.
2. Add 100 μl of 1x tissue removal mix per capture area and incubate for 40 minutes at 37℃.
3. Wash as follows:
   1. Wash the capture area with ultrapure water three times.
   2. Wash the capture area with 100 μl of freshly prepared 0.1N NaOH three times* (*each with 5 min incubation at room temperature).
   3. Wash the capture area with 100 μl of 0.1M Tris (pH7.5) three times.
   4. Wash the capture area with 100 μl of Ultrapure water three times.

Note

Visually confirm that tissue removal is complete after washes have been completed.

Second strand synthesis

Second strand synthesis mix

Reagent	Final concentration	Volume (μL)
NEBuffer-2	1x	10
100 uM randomer	10 uM	10
10 mM dNTPs	1 mM	10
Klenow exo (-) Fragment (5 U/uL)	0.5 U/uL	10
Ultrapure water		60
Total		100

1. Add 100 μL second strand synthesis mix per capture area. Incubate at 37°C for 2 h.
2. Wash with 100 μL ultrapure water 3 times.
3. Elute the second strand product by incubating the capture areas in 100 μl of freshly prepared 0.1 N NaOH twice for 5 min each. Recover the elutions and pool the second strand product together per sample.
4. Mix 200 μl of second strand product with 28.6 μl of 1M Tris-HCl pH 7.5. Proceed directly to next step.

Purify the 228.6 μL elution using AmpureXP beads at a ratio of 1.8 beads to 1x second strand product, following the manufacturer's instructions. Elute the product in 82.5 μL ultrapure water.

qPCR for cycle number assessment

Note

Use of a passive reference dye depends on the qPCR cycler used. We usually use the StepOne™ Real-Time PCR System (Applied Biosystems).

Pipette and run a qPCR to determine the appropriate cycle number to amplify your eluted second strands.

qPCR mix

Reagent	Final concentration	Volume (μl)
2x Blue S'Green qPCR mix + ROX	1x	10
10 uM Fw primer	1 uM	2
10 uM Rw primer	1 uM	2
Second strand product		2.5
Ultrapure water		3.5
Total		20

Temperature	Time	Cycles
95°C	3 min	1
95°C	30 sec	(40)
60°C	1 min	(40)
72°C	1 min	(40)

Derive the PCR cycle number required for the amplification of your sample as follows:

Set a threshold at 50% of the peak ΔRn. For each sample determine the cycle number at the intersection of the threshold and amplification curve. Subtract 5 cycles to account fo the qPCR input (3%). This number is your recommended PCR cycle number. We expect a cycling number between 11 and 14.

Library construction

Library amplification and purification

Library amplification mix

Reagent	Final concentration	Volume (μL)
2x KAPA HiFi Hotstart Readymix	1x	100
100 uM WTA1*F primer	1 uM	2
100 uM WTA1*R primer	1 uM	2
Purified 2nd strand		80
Ultrapure water		16
Total		200

Temperature	Time	Cycles
95°C	3 min	1
95°C	30 sec	(To be determined)
60°C	1 min	(To be determined)
72°C	1 min	(To be determined)
72°C	2 min	1
4°C	hold

1. Prepare the library amplification mix per sample.
2. Split each sample mix into four PCR tubes, each with 50 uL volume.
3. Run the PCR with the cycle number determined previously (3.9).
4. Pool the 200 μL PCR product per sample and purify using AmpureXP beads at a 1:1 ratio of beads PCR product, following the manufacturer's instructions.
5. Elute the PCR product in 20 μL ultrapure water.

Example BioAnalyzer profile before size selection

Size selection

Perform size selection of your sample to obtain fragments 350 - 1100 bp. Use the Bluepippin or PippinHT 1.5% agarose gel and follow the manufacturer's instructions. Measure the concentration of your size-selected product using the Qubit dsDNA quanitification kit.

Example BioAnalyzer profile after size selection