TITLE: BARseq - BARseq-styled in situ sequencing for barcoded rabies virus
AUTHORS: Xiaoyin Chen, Mara CP Rue

[DESCRIPTION]
This protocol describes the application of BARseq-style in situ sequencing adapted for barcoded rabies virus. Similar procedures for both trans-synaptic tracing and retrograde tracing experiments.

[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)
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3. Incubate for 1 hour at room temperature on slow shaker
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4. Wash the slides by immersing in 1x PBS (2 slides per 50ml falcon tube, back to back)
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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)
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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
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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 in and out of the chamber smoothly.
SECTION: Library preparation
12. Make reverse transription mix:  50 μM N20 primer (XC2757), 2 μM XCAI6, 2 μM XCAI7, 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. 

For the monosynaptic tracing experiments, the RT primers additionally included 2 μM of primers for the rabies glycoprotein (XCAI63 through XCAI76). 
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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
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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 non-gap-filling ligation mix: 1x Ampligase buffer, 20 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. 

In the retrograde tracing experiments, the non-gap-filling padlock probe mix included all padlock probes for endogenous genes. In the monosynaptic tracing experiments, the non-gap-filling padlock probe mix included all padlock probes for endogenous genes except Slc30a3, and additionally included padlocks for the rabies glycoprotein (XCAI77 – XCAI90).
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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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21. Wash twice in PBST
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26. 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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27. Incubate in RCA mix overnight at room temperature
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28. DAY 3:

Wash with PBST once
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29. 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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30. Wash with 1M Tris pH 8.0, then incubate in new 1M Tris pH 8.0 for 30 mins
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31. Wash twice in PBST
SECTION: Sequencing
32. Hybridization of Gene sequencing primer:

Wash with FISH wash (2x SSC with 10% formamide)
SECTION: Sequencing
33. Hybridize sequencing primer (YS220) with a primer concentration of 1 μM in FISH wash for 10 mins at room temperature
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34. Wash with FISH wash three times, 2 mins each
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35. Wash with PBST twice
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36. Sequence first cycle (genes and barcodes): 
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
36.1. Incorporation Buffer 60 °C 3 mins x1
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36.2. 2% PBST x1
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36.3. 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
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36.4. 2% PBST x1
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36.5. Incorporation Buffer  x2
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36.6. IRM  60 °C 3 mins x2
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36.7. 2% PBST x1
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36.8. 2% PBST  60 °C 3 mins x4
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36.9. Replace PBST with USM and Image  **if slides are dirty, clean with 70% Ethanol before adding USM** 
SECTION: Sequencing
37. Sequence subsequent cycles (genes and barcodes):
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
37.1. Incorporation buffer x2
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37.2. CRM 60 °C 3 mins x2
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37.3. Incorporation buffer x1 - Wipe ports after adding the incorporation buffer, to ensure that no CRM is left on the slide's surface
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37.4. 2% PBST x1
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37.5. 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
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37.6. 2% PBST x1
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37.7. Incorporation buffer x2
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37.8. IRM 60 °C 3 mins x2
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37.9. 2% PBST x1
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37.10. 2% PBST 60 °C 3 mins x4
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37.11. Replace 2% PBST with USM and image **if slides are dirty, clean with 70% Ethanol before adding USM** 
SECTION: Sequencing
38. After completing all gene sequencing imaging cycles: 

 Hybridization cycle
SECTION: Sequencing
38.3. 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
38.1. 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. 
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38.2. FISH wash (2x SSC with 10% formamide) 1x
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38.4. FISH wash x1 
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38.5. 0.002 mg/ML DAPI in PBST, room temperature for 5 mins
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38.6. Replace PBST with USM and image **if slides are dirty, clean with 70% Ethanol before adding USM** 
SECTION: Library preparation
20. Make the gap-filling ligation mix [same as the non-gap-filling mix with the rabies barcode padlock probe (XCAI5) as the only padlock probe, and with 50 μM dNTP, 0.2 U/μL Phusion DNA polymerase, and 5% glycerol]

1x Ampligase buffer, 20 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, 50 μM dNTP, 0.2 U/μL Phusion DNA polymerase, and 5% glycerol. 
SECTION: Library preparation
22. Incubate in ligation mix for 5 mins at 37 °C, then 45 mins at 45 °C. **exact timing on this step!**
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23. Wash twice in PBST, then once in FISH Wash (2x SSC with 10% formamide)
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24. Hybridize with 1 μM RCA primer (XC1417) in FISH wash for 10 mins at room temperature
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25. Wash twice in  FISH wash, then twice in PBST
SECTION: Sequencing
39. After completing all gene sequencing imaging cycles and hybridization cycle:

Hybridize barcode sequencing primers
SECTION: Sequencing
39.1. Strip buffer (60% formamide 2xSSC 0.01% Tween20) 
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. 
SECTION: Sequencing
39.2. FISH wash (2x SSC with 10% formamide) 1x
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39.3. Hybridize probe XCAI131 with a primer concentration of 1 μM in 2x SSC and 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
39.4. FISH wash x1 
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40. Sequencing barcodes: The sequencing procedures are the same as those for gene sequencing (Steps 36 and 37)

After hybridizing barcode primers, go directly back to step 36, sequence first barcode imaging cycle. Then repeat step 37 for desired length of barcode sequence.