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.

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)

Incubate for 1 hour at room temperature on slow shaker

Wash the slides by immersing in 1x PBS (2 slides per 50ml falcon tube, back to back)

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.

Wash twice in PBST (1x PBS + 0.5% Tween-20)

Wash in 70% Ethanol for 5 mins

Wash in 85% Ethanol for 5 mins

Wash in 100% Ethanol for 5 mins

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)

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.

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).

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.

DAY 2:

Wash with PBST once

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

Wash with 1M Tris pH 8.0, then incubate in new 1M Tris pH 8.0 for 30 mins

Wash twice in PBST

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).

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).

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.

Wash twice in PBST

Incubate in ligation mix for 5 mins at 37 °C, then 45 mins at 45 °C. exact timing on this step!

Wash twice in PBST, then once in FISH Wash (2x SSC with 10% formamide)

Hybridize with 1 μM RCA primer (XC1417) in FISH wash for 10 mins at room temperature

Wash twice in FISH wash, then twice in PBST

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

Incubate in RCA mix overnight at room temperature

DAY 3:

Wash with PBST once

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

Wash with 1M Tris pH 8.0, then incubate in new 1M Tris pH 8.0 for 30 mins

Wash twice in PBST

Hybridization of Gene sequencing primer:

Wash with FISH wash (2x SSC with 10% formamide)

Hybridize sequencing primer (YS220) with a primer concentration of 1 μM in FISH wash for 10 mins at room temperature

Wash with FISH wash three times, 2 mins each

Wash with PBST twice

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:

http://www.illumina.com/products/by-type/sequencing-kits/cluster-gen-sequencing-reagents/miseq-reagent-kit-v2.html

Incorporation Buffer 60 °C 3 mins x1

2% PBST x1

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

2% PBST x1

Incorporation Buffer x2

IRM 60 °C 3 mins x2

2% PBST x1

2% PBST 60 °C 3 mins x4

Replace PBST with USM and Image if slides are dirty, clean with 70% Ethanol before adding USM

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.

Incorporation buffer x2

2% PBST 60 °C 3 mins x4

Replace 2% PBST with USM and image if slides are dirty, clean with 70% Ethanol before adding USM

CRM 60 °C 3 mins x2

Incorporation buffer x1 - Wipe ports after adding the incorporation buffer, to ensure that no CRM is left on the slide's surface

2% PBST x1

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

2% PBST x1

Incorporation buffer x2

IRM 60 °C 3 mins x2

2% PBST x1

After completing all gene sequencing imaging cycles:

Hybridization cycle

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.

FISH wash (2x SSC with 10% formamide) 1x

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.

FISH wash x1

0.002 mg/ML DAPI in PBST, room temperature for 5 mins

Replace PBST with USM and image if slides are dirty, clean with 70% Ethanol before adding USM

After completing all gene sequencing imaging cycles and hybridization cycle:

Hybridize barcode sequencing primers

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.

FISH wash (2x SSC with 10% formamide) 1x

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.

FISH wash x1

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.