TITLE: BARseq  - high-throughput cell typing with in situ sequencing
AUTHORS: Xiaoyin Chen, Anthony M. Zador, Mararue

[DESCRIPTION]
This protocol describes the application of BARseq as a standalone in situ sequencing method to achieve multiplexed interrogation of endogenous genes. In this variation, BARseq is similar to in situ sequencing (ISS), but uses Illuimna SBS for sequencing readout.

[GUIDELINES]
Standard precautions with RNA samples should be taken to reduce RNA degradation during tissue processing and library preparation. Pipetting and suctioning should be gentle throughout the whole procedure, and sample should not be left dried.

[STEPS]
SECTION: Library preparation
1. Tissues with barcoded neurons should be cryo-sectioned to 20 μm and mounted on slides. Slides can be stored at -80 °C for up to a month.
SECTION: Library preparation
2. DAY 1

Take slide(s) out of -80 °C and immerse immediately in 4% paraformaldehyde in 1x PBS (2 slides per 50mL falcon tube, back-to-back)
SECTION: Library preparation
3. Incubate for 1 hour at room temperature on slow shaker
SECTION: Library preparation
4. Wash the slides by immersing in 1x PBS (2 slides per 50ml falcon tube, back to back)
SECTION: Library preparation
5. Wipe excess PBS off the surface of the chamber, then stick on the Hybriwell-FL chambers. Note that the ports on the chamber should be placed as far away from the tissue slices as possible. 
SECTION: Library preparation
6. Wash twice in PBST (1x PBS + 0.5% Tween-20)
SECTION: Library preparation
7. Wash in 70% Ethanol for 5 mins
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8. Wash in 85% Ethanol for 5 mins
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9. Wash in 100% Ethanol for 5 mins
SECTION: Library preparation
10. Replace with new 100% Ethanol, drop extra 100% Ethanol on top of slides and cover with ParaFilm to avoid evaporation. Incubate for at least 1.5 hrs at 4 °C (up to 3 hours)
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11. Wash in PBST for 4-6 times, until all bubbles are cleared in the chamber and PBST flows into and out of the chamber smoothly. 
SECTION: Library preparation
12. Make reverse transription mix:  50 μM N20 primer (XC2757), 20 U/μL RevertAid H Minus M-MuLV reverse transcriptase, 500 μM dNTP, 0.2 μg/μL BSA, 1 U/μL RiboLock RNase Inhibitor, 1x RevertAid RT buffer. 
SECTION: Library preparation
13. Incubate in reverse transcription mix overnight at 37 °C. Create a humidity chamber to avoid the slides drying out using kim-wipes and DI water. 
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14. DAY 2:

Wash with PBST once
SECTION: Library preparation
15. Incubate in a mixture of 1μL BS(PEG)9 per 4 μL PBST (e.g. 200ul BS(PEG)9 and 800ul PBST) for one hour at room temperature
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16. Wash with 1M Tris pH 8.0, then incubate in new 1M Tris pH 8.0 for 30 mins
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17. Wash twice in PBST
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18. Make ligation mix: 1x Ampligase buffer, 100 nM padlock probe each, 0.5 U/μL Ampligase, 0.4 U/μL RNase H, 1 U/μL RiboLock RNase Inhibitor, 50 mM KCl (extra of those already provided by the ampligase buffer), 20% formamide.
SECTION: Library preparation
19. Incubate in ligation mix for at least 30 mins at 37 °C (can go longer but not shorter), then at least 45 mins at 45 °C (can go longer but not shorter).
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20. Wash twice in PBST
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21. Make RCA mix: 1 U/μL phi29 DNA polymerase, 1x phi29 polymerase buffer, 0.25 mM dNTP, 0.2 μg/μL BSA, 5% glycerol (extra of those from the enzymes), 125 μM aminoallyl dUTP
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22. Incubate in RCA mix overnight at room temperature
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23. DAY 3:

Wash with PBST once
SECTION: Library preparation
24. Incubate in a mixture of 1μL BS(PEG)9 per 4 μL PBST (e.g. 200ul BS(PEG)9 and 800ul PBST) for one hour at room temperature
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25. Wash with 1M Tris pH 8.0, then incubate in new 1M Tris pH 8.0 for 30 mins
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26. Wash twice in PBST
SECTION: Sequencing
27. Hybridization of Gene sequencing primer:

Wash with FISH wash (2x SSC with 10% formamide)
SECTION: Sequencing
28. Hybridize sequencing primer (YS220) with a primer concentration of 1 μM in FISH wash for 10 mins at room temperature
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29. Wash with FISH wash three times, 2 mins each
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30. Wash with PBST twice
SECTION: Sequencing
31. Sequence first cycle:
Do the following incubations. Unless noted with incubation temperature, each step is performed at room temperature. For steps without incubation time, treat these as quick washes. large flat metal blocks can be used to place sample slides to quickly cycle through high and low temperatures. This version uses MiSeq Nano v2 kit:
https://www.illumina.com/products/by-type/sequencing-kits/cluster-gen-sequencing-reagents/miseq-reagent-kit-v2.html
SECTION: Sequencing
31.1. Incorporation Buffer 60 °C 3 mins x1
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31.2. 2% PBST x1
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31.3. Idoacetamide blocker: For 9.3mg vial dilute pellet in between 2.5-3.5mL 2% PBST. Make fresh tube daily and store out of light. 

Idoacetamide blocker 60 °C 3 mins x1
SECTION: Sequencing
31.4. 2% PBST x1
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31.5. Incorporation Buffer  x2
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31.6. IRM  60 °C 3 mins x2
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31.7. 2% PBST x1
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31.8. 2% PBST  60 °C 3 mins x4
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31.9. Replace 2% PBST with USM and Image **if slides are dirty, clean with 70% Ethanol before adding USM** 
SECTION: Sequencing
32. Sequence subsequent cycles:Do the following incubations. Unless noted with incubation temperature, each step is performed at room temperature. For steps without incubation time, treat these as quick washes. large flat metal blocks can be used to place sample slides to quickly cycle through high and low temperatures. 
SECTION: Sequencing
32.1. Incorporation buffer x2
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32.2. CRM 60 °C 3 mins x2
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32.3. Incorporation buffer x1 - Wipe ports after adding the incorporation buffer, to ensure that no CRM is left on the slide's surface
SECTION: Sequencing
32.4. 2% PBST x1
SECTION: Sequencing
32.5. Idoacetamide blocker: For 9.3mg vial dilute pellet in between 2.5-3.5mL 2% PBST. Make fresh tube daily and store out of light. 

Idoacetamide blocker 60 °C 3 mins x1
SECTION: Sequencing
32.6. 2% PBST x1
SECTION: Sequencing
32.7. Incorporation buffer x2
SECTION: Sequencing
32.8. IRM 60 °C 3 mins x2
SECTION: Sequencing
32.9. 2% PBST x1
SECTION: Sequencing
32.10. 2% PBST 60 °C 3 mins x4
SECTION: Sequencing
32.11. Replace 2% PBST with USM and image **if slides are dirty, clean with 70% Ethanol before adding USM** 
SECTION: Sequencing
33. Hybridization cycle
SECTION: Sequencing
33.3. Hybridize probes (YS221, XC2758, XC2759, XC2760) with a primer concentration of 1 μM in FISH wash at 60 ˚C for 2 minutes, then for 10 mins at room temperature. Rotate plates in holder to ensure they cool down slowly. 

SECTION: Sequencing
33.1. Hybridize probes:
Make strip buffer: 60% formamide 2xSSC 0.01% Tween20

Strip buffer 60 ˚C 5 mins x3 
Cool down quickly on metal plates between washes, place on metal plates in 60 ˚C oven to heat up quickly. 
33.8.    
33.7.  
SECTION: Sequencing
33.2. FISH wash (2x SSC with 10% formamide) 1x
SECTION: Sequencing
33.4. FISH wash x1 
SECTION: Sequencing
33.5. 0.002 mg/ML DAPI in 2% PBST, room temperature for 5 mins
SECTION: Sequencing
33.6. Replace PBST with USM and image **if slides are dirty, clean with 70% Ethanol before adding USM**