Jun 27, 2025

Public workspaceBARseq 2.5

DOI
https://dx.doi.org/10.17504/protocols.io.kqdg3ke9qv25/v1
BARseq 2.5
  • Jonathan Wang1,
  • Yoh Isogai1
  • 1Allen Institute for Neural Dynamics
  • Allen Institute for Neural Dynamics
Jonathan Wang
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DOI: https://dx.doi.org/10.17504/protocols.io.kqdg3ke9qv25/v1
Protocol Citation: Jonathan Wang, Yoh Isogai 2025. BARseq 2.5. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg3ke9qv25/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 17, 2025
Last Modified: June 27, 2025
Protocol Integer ID: 124544
Keywords: BARseq, In Situ Sequencing, Spatial Transcriptomics, In Situ Hybridization , AAV, Cell Type , barcoded gene, profiling enhancer aav, situ transcriptomics method, high throughput in situ transcriptomics method, amplification process for multiplexed gene detection, multiplexed gene detection, detecting endogenous gene transcript, barseq, enhancer aav, significant marker genes of interest, significant marker gene, designed barcode, endogenous gene transcript, identifying cell type, transcript, such as transcript, sequencing, situ probe hybridization, specific cell type
Funders Acknowledgements:
Allen Institute
Grant ID: ___
Abstract

BARseq is a high throughput in situ transcriptomics method utilizing designed barcodes incorporated in the amplification process for multiplexed gene detection. Readout methods include: 1) In situ sequencing detecting endogenous gene transcripts for identifying cell types at subclass level; 2) in situ sequencing of exogenous barcoded gene, such as transcripts derived from Sindbis virus; 3) In situ probe hybridization directly labeling 1-4 significant marker genes of interest.

BARseq2.5 Protocol is an updated version of BARseq2.0 with an overall higher detection sensitivity and quality. At the Allen Institute For Neural Dynamics, this protocol is implemented for the purpose of profiling enhancer AAVs designed to target specific cell types of interest. Although not specifically included in this protocol, BARseq2.5 has also been used for other purposes such as axonal projection mapping.

Guidelines
  • Unsealed, sectioned samples kept more than 18 months in freezer is not recommended for BARseq as endogenous gene readout yields poor signals and lower spot counts.

  • This protocol does not include the sequencing of axonal projection barcodes. However there is no conflict to incorporate that part of the protocol (as described in BARseq2.0 protocol) into BARseq2.5 and will work as usual.

  • Volume of various reaction mixtures depends on the numbers of slides running the experiment, this protocol uses 4 slides (8 chambers) as an example.

  • For the rest of this protocol, a single wash in and out of chambers is indicated in syntax as such: 0.5% PBST Wash ; 5 sec ; 25 °C. For this example it means to pipette 0.5% PBST into chamber and immediately suction out of the chamber after 5 seconds sitting on an aluminum block at room temperature. Pipetting and suctioning should be as gentle as possible to prevent sample being left dried.
Materials

Note
Any materials used in this protocol not listed specifically implies using any generic brand/type of the item likely doesn't have significant effect on reproducibility.

Reagents

Reagent20% PFA - 20% Paraformaldehyde (formaldehyde) aqueous solution, 10x10 mLElectron Microscopy SciencesCatalog #15713
ReagentNuclease-Free Water (not DEPC-Treated)Thermo Fisher ScientificCatalog #AM9932
ReagentNuclease-Free Water (not DEPC-Treated) 100 mLThermo Fisher ScientificCatalog #AM9938
ReagentKCl (2 M), RNase-freeThermo Fisher ScientificCatalog #AM9640G
ReagentPBS - Phosphate-Buffered Saline (10X) pH 7.4, RNase-freeThermo Fisher ScientificCatalog #AM9625
ReagentSSC (20X), RNase-freeThermo Fisher ScientificCatalog #AM9763
ReagentTWEEN® 20Merck MilliporeSigma (Sigma-Aldrich)Catalog #P1379
ReagentTris-HCl (1 M), pH 8.0, RNase-freeThermo Fisher ScientificCatalog #AM9856
ReagentDimethyl sulfoxideMerck MilliporeSigma (Sigma-Aldrich)Catalog #276855
ReagentInvitrogen™ Formamide (Deionized)Thermo Fisher ScientificCatalog #AM9342 (AM9344)
ReagentRevertAid H Minus Reverse Transcriptase (200 U/μL)Thermo Fisher ScientificCatalog #EP0452
Note
Including:
  • RT (Reverse Transcriptase)
  • RT Buffer (Reverse Transcriptase Buffer)

ReagentRiboLock RNase Inhibitor (40 U/μL)Thermo Fisher ScientificCatalog #EO0381
ReagentRecombinant Albumin, Molecular Biology GradeNew England BiolabsCatalog #B9200S
ReagentRNase H (5000 U)QiagenCatalog #Y9220L
ReagentAmpligase Thermostable DNA LigaseLGC biosearchCatalog #A0102K
ReagentAmpligase 10X Reaction bufferLGC biosearchCatalog #A1905B
ReagentAminoallyl-dUTP Solution (50 mM)Thermo Fisher ScientificCatalog #R1101
Reagentphi29-XT RCA KitNew England BiolabsCatalog #E1603L

Note
Including:
  • Phi29XT polymerase
  • Phi29XT polymerase buffer
  • dNTP

Reagentphi29 DNA Polymerase (10 U/μL)Thermo Fisher ScientificCatalog #EP0094

Note
Including:
  • Phi29 polymerase
  • Phi29 polymerase buffer

ReagentIodoacetamide, No-Weigh™ FormatThermo Fisher ScientificCatalog #A39271
ReagentMiSeq Reagent Nano Kit v2 (300-cycles)Illumina, Inc.Catalog #MS-103-1001

Note
Including:
  • USM (SRE) - Imaging/scan Mix
  • IRM (IMS) - Incorporation Mix
  • CRM (CMS) - Cleavage Mix
  • Incorporation buffer

ReagentDAPI (4,6-diamidino-2-phenylindole, dihydrochloride)Thermo Fisher ScientificCatalog #D1306
ReagentBS(PEG)9Thermo Fisher ScientificCatalog #21582
ReagentGlycerolMerck MilliporeSigma (Sigma-Aldrich)Catalog #G6279
ReagentEthyl AlcoholThermo Fisher ScientificCatalog #UN1170
ReagentdNTP Mix (10 mM each) (deoxynucleotide triphosphate mixtures containing dATP, dCTP, dGTP and dTTP)Thermo Fisher ScientificCatalog #R0194

Other Consumables

ReagentHybriWell™ Sealing System - Hybridization Cover - HBW2222FLGrace Bio-LabsCatalog #611204
ReagentFisherbrand™ Tissue Path Superfrost™ Plus Gold Slides 144/PKThermo Fisher ScientificCatalog #15-188-48
ReagentThermo Scientific™ RNase AWAY™ Surface DecontaminantThermo Fisher ScientificCatalog #21-402-178
ReagentFalcon® 50 mL High Clarity PP Centrifuge Tube Conical Bottom Sterile 25/Rack 500/CaseCorningCatalog #352098, 430776, 431123
ReagentKimberly-Clark Professional™ Kimtech Science™ Kimwipes™ Delicate Task Wipers, 1-PlyFisher ScientificCatalog #06-666A
ReagentStart Kit PL-LTS 2,20,200,1000μL 30386597 - Pipette (Mettler-Toledo Rainin, LLC)RaininCatalog #30386597
ReagentParafilm® M Sealing FilmVWR International (Avantor)Catalog #52858-000

Equipment

ReagentVactrap™ 2 Vacuum Trap System 2 1 L HDPE Bottle Assembly with Tray, 0.2 µm Vent Filter, Not AutoclavVWR International (Avantor)Catalog #76207-602
ReagentRapidFISH Slide Hybridizer 115VBoekel ScientificCatalog #240200 (40 °C) (45 °C)
ReagentSureTemp ™ Dual-Convection Incubators, 40LBenchmark ScientificCatalog #H2505-40 (37 °C)
ReagentUVP HB-1000 Hybridization IncubatorThermo Fisher ScientificCatalog #UVP95003001 (60 °C)
ReagentStainTray™ - Slide Staining System - 10 Slide Staining Tray With Black LidElectron Microscopy SciencesCatalog #71396-B
ReagentVWR® Modular Heating Blocks, Solid - Double 15.1 x 9.5 x 5.7 cmVWR International (Avantor)Catalog #13259-297
ReagentVWR® Standard 1000 Standard Orbital ShakerVWR International (Avantor)Catalog #89032-088

PPE
  • Gloves
  • Mask
  • Lab Coat

Microscope

Nikon inverted research microscope ECLIPSE Ti2-E/Ti2-E/B

Table 1. Laser and filter settings used for imaging the endogenous sequencing cycles

Table 2. Laser and filter settings used for imaging the hybridization cycle


Troubleshooting
Safety warnings
General PPE required (Gloves, Lab coat). Extra Caution When Handling The Following Liquids:


Before start
Start Day 1 from carrying initial processing of sample slices already mounted onto slides stored in a -80°C freezer.


Library Preparation Day 1
Initial Preparation
Working environment must be RNase free until Day 2 where mRNA has been reverse transcribed into more stable cDNA.

Wearing a mask throughout Day 1 is highly recommended to minimize mRNA degradation of samples
Clean lab bench/workstation area with RNase Away spray
Regularly replace pipette tips attached to vacuum system
Fixation
Make 4% Paraformaldehyde (PFA) in 1X Phosphate Buffered Saline (PBS) in a 50mL sterile, conical tube (Need 1 tube for every 2 slides)
Make 1X PBS in a 50mL sterile, conical tube (Need 1 tube for every 2 slides)
Fetch slides from -80 °C freezer
Immerse slides immediately in 4% PFA (2 slides positioned back to back in each tube)
Place tube with slides on a shaker at low speed (~80 rpm), make sure the slides inside are sitting horizontally to avoid bubbles
Incubate for 1 hour at room temperature (20-25 °C)
Use forceps to transfer slides from 4% PFA into 1X PBS (full immersion the same way to stop reaction)
Dispose the 4% PFA into a dedicated liquid waste
Chamber Installation
The goal of this step is to place 2 HybriWell covers onto each slide to create chambers for further chemistry to take place.


Vacuum setup: insert a pipette tip (no filter) into a vacuum line
Make 0.5% Phosphate Buffered Saline with Tween (PBST) in a 50mL sterile, conical tube (Used throughout library preparation)
Use forceps to take out the slides from the PBS tube
Dry the back of the slide by placing it on a Kimwipe
Dry the edges/perimeter around the sections of the front of the slide with another folded Kimwipe
Take out 2 Grace bio-lab hybriwell-FL (hybriwell) covers per slide
For each slide, pair up every 2 hybriwells, touching side by side on the table
Peel off the sticker (thin film) of the hybriwell
Hold up the slide horizontally with the label part of the slide on your left hand
Have the slide facing down towards the table, and directly stick onto the hybriwells. Press around the edges from the slide side to make sure good contact.
Pipette/inject 0.5% PBST via the lower right pin hole of the hybriwells into the chamber until it is full (~150 uL)
Dehydration
Make 70% Ethanol in a 50mL sterile, conical tube
Make 85% Ethanol in a 50mL sterile, conical tube
* Optional to make 1mL which is sufficient for 4 slides
Pick up the slide with one hand, press finger on the transparent tap, vacuum out the 0.5% PBST
Pipette 0.5% PBST again for a second wash, set it aside and move onto the next step
Fetch a slide holder box, clean the holder box using RNase away spray bottle, set it aside
Vacuum out the 0.5% PBST
70% Ethanol Wash ; 5 min ; 25 °C
85% Ethanol Wash ; 5 min ; 25 °C
100% Ethanol Wash ; 5 min ; 25 °C
Inject 100% ethanol into the chamber
Add a drop or two of the 100% ethanol onto the surface of the hybriwell (chamber)
Cut out 1 rectangular piece of parafilm for each slide
Gently place parafilm on top of the chambers
Place the slides into the slide holder box
Note
It's important to keep holder box dry. Optionally, seal the slide holder box in a plastic bag or place Kimwipes sprayed with 100% Ethanol inside the box. Do not use any water or other liquid to wet Kimwipes that will add moisture.

Place the holder box in the 4C fridge and let it incubate for at minimum of 1.5 hours (up to 3 hrs max)
Reverse Transcription
Reverse transcribe mRNA to generate more stable cDNA (Template broken down with RNase H on Day 2 after crosslinking)
Schematics of reverse transcription
Overnight
Thaw the following: Primers (ordered custom), RT Buffer, dNTP


Take out the slide box from the 4 °C fridge
Vacuum out the 100% ethanol
Pipette 0.5% PBST into the chamber, let it sit for 5 minutes
Vacuum out half of the 0.5% PBST, and then refill, let it sit for 5 minutes
Vacuum out the 0.5% PBST
Repeat 0.5% PBST Wash ; 5 sec ; 25 °C for 3~5 times until liquid can enter/exist chamber smoothly
Pipette 0.5% PBST, set it aside and move onto the next step
Prepare RT Mix in a RNAse-free microcentrifuge tube in the following concentration and order:
The following reagents should be kept in a benchtop cooler or in dry ice when taken out of the freezer: BSA, Ribolock RNase Inhibitor, RevertAid H Minus Reverse Transcriptase. Flick and spin down the solution. Avoid vortexing the enzyme mix.

Vacuum out the 0.5% PBST
Carefully Pipette the RT mix into the chambers
Place the slides back into the holder box
Fold four Kimwipes and place in the holder box
Add ~7.5 mL of water into the slide holder box to fully wet the Kimwipes for humidification
Place the entire holder box into a 37 °C oven, incubate for +12 hours overnight
Library Preparation Day 3
5h 45m
Fixate cDNA
Be careful not to over suction until section dried out and appears white. To avoid drying out the section, vacuum liquid from chambers slowly by pressing one finger on the transparent tab of the Hybriwell to control flow rate.

Thaw BS(PEG)9 Crosslinker

Note
*Please note that BS(PEG) can occasionally differ from batch to batch, in which could affect experimental outcome (i.e. blurry signal)

Add 460 uL of anhydrous DMSO (Dimethyl Sulfoxide) into 100 mg of BS(PEG)9 vial

Note
Any excess dissolved BS(PEG)9 can be stored in -20 °C freezer. Seal the vial with parafilm, and place in a desiccator. Opened BS(PEG)9 should not be used if stored more than a week.

Take out slides from 37 °C oven, vacuum RT mixture and replace with 0.5% PBST
Prepare the following reaction mixture:
Carefully vacuum 0.5% PBST from chamber
Pipette the crosslinker mixture in, place slides back into the holder box with the lid on
Incubate at room temperature for Duration01:00:00

1h
Vacuum cross linker mix, inject 1M Tris-HCl (pH 8.0) into the chambers to stop reaction
Place slide back into the holder box
Incubate for at least Duration00:30:00 at room temperature (can incubate up to 2 hours maximum)
Padlock Annealing
Anneal designed padlocks (carrying barcode sequence) targeting genes of interest. Ampligase is used for ligating the padlock to form a complete loop as template for the rolling circle amplification (RCA) (Step 8)
Schematics of Padlock hybridization and DNA ligation
Thaw the following: all padlocks (ordered custom), ampligase buffer, formamide
Prepare the following padlock mix for example:
The following reagents should be kept in a benchtop cooler or in dry ice when taken out of the freezer: Ampligase, Ribolock RNase Inhibitor, RNase H.
Set the mixture aside, vacuum Tris-HCl from the chambers, and replace with 0.5% PBST


Repeat with another 0.5% PBST Wash ; 5 sec ; 25 °C
Remove 0.5% PBST, inject chambers with the mixture
Place slide back into the holder box
Transfer the entire holder box inside a 37 °C oven
Incubate at 37 °C for exactlyDuration00:30:00

30m
Immediately transfer the slides into a 45 °C hyb oven
Continue the incubation at 45 °C for Duration00:45:00 (can last up to 1 hour)

Note
Start preparing the RCA mix (next step) 15 minutes before the 45 °C incubation is about to end. Prolonged time interval between step 7 and step 8 could lead to poor signal quality.
45m
Rolling Circle Amplification
Amplify copies of the padlock into rolonies for signal detection purposes. The RCA is a two step incubation using polymerase phi29XT followed by phi29.
Schematics of rolling circle amplification
Overnight
Thaw the following: dNTP (phi29XT Kit), phi29XT polymerase buffer
Prepare the phi29XT polymerase reaction mix as follows:

The following reagents should be kept in a benchtop cooler or in dry ice when taken out of the freezer: Phi29XT polymerase
Take slides out of the oven, remove the padlock mix
0.5% PBST Wash ; 5 sec ; 25 °C (Repeat 3 times)
Pipette the phi29XT reaction mix into the chamber
Transfer slides into 40 °C oven, let it incubate for Duration02:00:00

2h
Thaw the following: dNTP, aadUTP, phi29 polymerase, phi29 polymerase buffer
Prepare phi29 reaction mix as follows:
The following reagents should be kept in a benchtop cooler or in dry ice when taken out of the freezer: BSA, Phi29 polymerase
Vacuum out the previous phi29XT mix, and inject the phi29 mix
Place slide back into the holder box
Incubate at room temperature overnight (>12 hours)
Library Preparation Day 3
5h 45m
Fixation
Final fixation for securing the RCA products in place using BS(PEG)9
Thaw BS(PEG)9 Crosslinker
If BS(PEG)9 stock has not been prepared, perform the following step:
Add 460 uL of DMSO into 100 mg of BS(PEG)9 vial


Prepare the following reaction mixture:

Vacuum out the phi29 mix from chamber and replace with 0.5% PBST
Vacuum out the 0.5% PBST, inject the reaction mix into chambers
Place slides back into the holder box
Incubate at room temperature for Duration01:00:00

1h
Stop reaction by vacuuming out the reaction mix and inject 1M Tris-HCl (pH 8.0) into chambers
Incubate at room temperature for Duration00:30:00 (can incubate up to 2 hours maximum)

30m
Storage

Vacuum out Tris-HCl from chambers

Inject 0.5% PBST into the chambers
Store slides in 4°C fridge before use (Ideally < 1 week before moving onto the next step for imaging)DurationOvernight

Hybridization Cycle [1 Cycle]
15m
Reagents Preparation
Thaw the following: USM, DAPI stock solution, all Hyb labeling probes
Make FISH Wash (10% Formamide) in a sterile, conical 50mL tube (opaque or covered with aluminum foil)
Note: FISH (fluorescent in-situ hybridization) Wash is
Make 1:500 DAPI diluted working solution in a 1.5 mL microcentrifuge tube

Labeling
This step directly labels the presence of enhancer AAV transcripts and 1-4 optional marker genes of interest.
Note
  • For the rest of this protocol, a single wash in and out of chambers is indicated in syntax as such: 0.5% PBST Wash ; 5 sec ; 25 °C. For this example it means to pipette 0.5% PBST into chamber and immediately suction out of the chamber after 5 seconds sitting on a aluminum block at room temperature. Pipetting and suctioning should be as gentle as possible to prevent sample being left dried.

  • During gene sequencing, slides must be placed immediately onto an aluminum block for quick cooling down back to room temperature after any heating incubation, unless specified otherwise. In situ sequencing uses reagents from illumina MiSeq kit containing: IRM (IMS), CRM (CMS), USM (SRE), & incorporation buffer.

  • The following example uses 4 hybridization oligonucleotides, but this should be adjusted to each user's needs.

Schematics of fluorophore conjugated probe hybridization
Imaging
Vacuum out 0.5% PBST and pipette FISH wash into the chambers
Set slides aside, move onto preparing the labeling mix:

Vacuum out FISH wash and inject the labeling mixture in
Incubate slides in 60°C UVA Hyb oven for Duration00:02:00 on heat block

2m
Place slides inside a slide box with chamber. Ensure the slides do not touch anything to allow for air-cooling
With the slide box lid on, let slides incubate at room temperature for Duration00:10:00 in the dark

10m
FISH Wash ; 5 sec ; 25 °C
Pipette diluted 1:500 DAPI working solution into chambers
Let slide incubate in the slide box in dark at room temperature for Duration00:03:00

3m
Vacuum out DAPI and wipe both sides of the slides using Kimwipe with a bit of ethanol
Pipette USM into the chambers
Image slides
Strip [Between Hybridization & Sequencing Cycles]
30m
Reagents Preparation
Make Strip Buffer in a 50mL sterile conical tube (opaque or covered with aluminum foil)
Stripping
15m
Vacuum USM from chambers

Strip Buffer ; 5 min ; 60 °C (UVA Hyb Oven) Duration00:05:00 (Repeat 3 times)
15m
Vacuum out strip buffer and add PBST 0.5%
Place the slides under the microscope to check if previous signals are gone, if not repeat 1x more wash
Gene Sequencing Cycle [7 Cycles]
12m
Reagents Preparation
Make 0.5% PBST in a 50mL sterile conical tube

Make 2% PBST in a 50mL sterile conical tube

Make FISH Wash in a 50 mL sterile conical tube (opaque or covered with aluminum foil)
Add 3 mL of 2%PBST to reconstitute Iodoacetamide in a 5 mL microcentrifuge tube
Store solution away from light. Make 3 mL of fresh Iodoacetamide dissolved in 2% PBST for every 12 hours. Don't use any dissolved Iodoacetamide stored overnight, Iodoacetamide is reactive and unstable when dissolved.
Thaw the following: USM, IRM, CRM, sequencing primer (YS220. Custom, ordered form IDT)
Primer Annealing
Vacuum out previous 0.5% PBST from the chambers
0.5% PBST ; 5 sec ; 25 °C

FISH Wash ; 5 sec ; 25 °C
Set slides aside, move onto preparing the sequencing primer working solution as follows:

Place slides inside the 60 °C UVA Hyb Oven and incubate for Duration00:02:00 on heat block

2m
Place slides inside a slide box with chamber. Ensure the slides do not touch anything to allow for air-cooling
With the slide box lid on, let slides incubate at room temperature for Duration00:10:00 in the dark

10m
Initial Cycle Sequencing (cycle 1)
In situ sequencing - This section of steps is to simultaneously detects multiple endogenous gene transcripts via 7 rounds of sequencing and imaging the barcodes, utilizing the MiSeq Reagent Nano Kit v2 (300-cycles) from Illumina.
Schematics of in situ sequencing
Imaging
Incorporation Buffer ; 3 min ; 60 °C
2% PBST ; 5 sec ; 25 °C
Iodoacetamide ; 3 min ; 60 °C
2% PBST ; 5 sec ; 25 °C
Incorporation Buffer ; 5 sec ; 25 °C (Repeat 2 times)
IRM ; 3 min ; 60 °C (Repeat 2 times)
2% PBST ; 5 sec ; 25 °C
2% PBST ; 3 min ; 60 °C (Repeat 4 times)
Wipe both sides of the slides using Kimwipe with a bit of ethanol
Pipette USM into the chambers
Image slides
Subsequent Cycles Sequencing (Repeat this step for cycle 2,3,4,5,6,7)
Barcode is 7 nucleic bases long. The 8th base is "C" for all barcode, thus it can be sequenced up to 8th cycle in which should give signal only in the "C" channel when imaging as a measure to confirm that each sequencing cycle was performed at high efficiency.

Imaging
Incorporation Buffer ; 5 sec ; 25 °C (Repeat 2 times)
CRM ; 3 min ; 60 °C (Repeat 2 times)
Incorporation Buffer ; 5 sec ; 25 °C
2% PBST ; 5 sec ; 25 °C
Iodoacetamide ; 3 min ; 60 °C
2% PBST ; 5 sec ; 25 °C
Incorporation Buffer ; 5 sec ; 25 °C (Repeat 2 times)
IRM ; 3 min ; 60 °C (Repeat 2 times)
2% PBST ; 5 sec ; 60 °C (Repeat 4 times)
Wipe both sides of the slides using Kimwipe with a bit of ethanol
Pipette USM into the chambers
Image slides
Protocol references
Version:
BARseq Version 2.0 (Xiaoyin Chen, Anthony M. Zador, Mara Rue)


Citation:
Xiaoyin Chen, Anthony M. Zador, Mararue 2023. BARseq - high-throughput cell typing with in situ sequencing. protocols.iohttps://dx.doi.org/10.17504/protocols.io.81wgbp4j3vpk/v2Version created by Xiaoyin Chen





























Figures Created in BioRender. W, J. (2025) https://BioRender.com/63mitxi
Acknowledgements
We would like to thank Xiaoyin Chen and Mara Rue for their help in setting up BARseq experiments.