RT-free Protocol

2023.6.2

*The reagent amount used in chip depends on the sample area, for half mouse brain, 50ul is recommended.

you need to ensure the sample is fully immersed in reagent!

1. fixation of the slides (~60min)

i. Incubate the slides in 200ul 0.1% GA (戊二醛)in 4% PFA (freshly prepared) for 15-30 min at RT 1µL 50% GA + 500 µL PFA

ii. Wash the slides in 200ul 1 x 2 min PBS-Tween 0.05%

iii. Wash the slides in 200ul 80% EtOH

iv. Assemble the PDMS chamber to glass slide

v. Wash the slides in 50 ul 1 x 2 min PBST

Permeabilization of the tissue (~10min)

i. Incubate the slides in 0.04% pepsin in 0.1M HCl at 37C for 2 min (在四度冰箱)

ii. Wash the slides for 1 x 2 min with PBS-Tween 0.05%

3. Dehydration (~20min)

i. Incubate the slides in 80% ETOH for 10 min

ii. Incubate the slides in 100% ETOH for 2 min

iii. wash the slides for 3 x 2 min PBS-Tween 0.05% for rehydration.

4. Blocking (30min)

Reagents	stock	final	1 x slide
Rnase Free H₂O
Ampligase Buffer	10x	1x	10μl
KCl	1M	0.05M	5μl
Formamide deionized	100%	20%	20μl
Probes oligo	10μM	0.05μM	1μl
BSA	10μg/μl	0.2μg/μl	2μl
RiboLock (Thermo)	40U/μl	1U/μl	2.5μl
tRNA (Ambion AM7119)	10μg/μl	0.2μg/μl	2μl
Total			100μl

i. Add the blocking solution into the chamber, mix by gentle pipetting (3-5 times) and incubate the slides at room temperature (RT) for 30 min.

5. Hybridization of Padlock probes (3h)

Reagents	stock	final	1 x slide
Rnase Free H₂O
Ampligase Buffer	10x	1x	10μl
KCl	1M	0.05M	5μl
Formamide deionized	100%	20%	20μl
Probes 0.5 μl/padlock	10μM	0.05μM	0.2μl
BSA	10μg/μl	0.2μg/μl	2μl
RiboLock (Thermo)	40U/μl	1U/μl	2.5μl
tRNA (Ambion AM7119)	10μg/μl	0.2μg/μl	2μl
Total			100μl

i. Add the solution into the chamber, mix by gentle pipetting (3-5 times) and seal.

ii. Incubate the slides at 55oC for 18 min for denaturation and at 45o C for 120 min for hybridization of the probes onto the target mRNA.(盖上盖子，烘箱温度可调成62-63和52-53).

iii. Wash the slides for 3 x 10 min with washing buffer (10% formamide in 2X SSC) to remove unhybridized probes. 100µL 20X SSC + 100µL formamide + 800µL H₂O.

iv. Wash the slides for 3 x 1 min with PBS-Tween 0.05% to remove the remaining formamide, as it can deactivate the enzyme in the following step.

6. Ligation of Padlock probes (~2h)

The ligation of the hybridized padlock probes is mediated by SplintR ligase (PBCV-1 DNA Ligase) which can function with DNA:RNA hybrid molecules.

Reagents	stock	final	1 x slide
RNase Free H₂O			75.5 ul
SplintR Buffer	10X	1X	10 ul
BSA	10 ug/ul	0.2 ug/ul	2 ul
SplintR (NEB)	25 U/ul	2.5 U/ul	10 ul
RiboLock (Thermo)	40 U/ul	1 U/ul	2.5 ul
Total			100 ul

i. Add the solution into the chamber, mix by gentle pipetting (3-5 times) and seal

ii. Incubate the slides at 37**o**C for 2 hours

iii. Wash the slides for 2 x 1 min with PBS-Tween 0.05%.

7. Rolling circle amplification (RCA, ~3h-17h)

Reagents	stock	final	1 x slide
RNase Free H₂O
Phi29 NEB	10uM	1uM	10ul
Phi29 Buffer	10X	1X	10 ul
Glycerol	100%	10%	10 ul
dNTPs	10 mM	0.25mM	2.5 ul
NH2-dUTP	2.5 mM	62.5 uM	2.5 ul
BSA	10 ug/ul	0.2 ug/ul	2 ul
RCA Primer 1+2	10uM for both	0.3uM for both	3 + 3 ul
Total			100 ul

i. Add the solution into the chamber, mix by gentle pipetting (3-5 times) and seal

ii. Incubate the slides at 30o C for 12-16 hours (For 2h, need to incubate at 37C)

iii. Wash the slides for 2 x 1 min with PBS-Tween 0.05%.

iv. Add 0.2% PEG9 in PBST mix by gentle pipetting (3-5 times) and incubate for 15-30 min at RT to fix the RCA products on the tissue.

2µL PEG9 BS + 98µL PBST

v. Wash the slides for 3 x 1 min with PBS-Tween 0.05%.

vi. Wash the slides for 3 x 2-10 min with 65% formamide at 30o C on a PCR machine block or an Eppendorf Thermostat. 650 µL Formamide + 350 µL H₂O

vii. Wash the slides for 2 x 1 min with PBS-Tween 0.05%

In total, this technology takes 16-24 h for library construction, 5h for 10 cyc sequencing rounds, taking the advantages of RT-free and in-situ SBS.