Aug 23, 2022

BICCN_DART-FISH

Nature Communications
DOI
https://dx.doi.org/10.17504/protocols.io.yxmvmnd45g3p/v1
  • 1University of California, San Diego
Chien-Ju Chen
Icon indicating open access to content
QR code linking to this content
DOI: https://dx.doi.org/10.17504/protocols.io.yxmvmnd45g3p/v1
External link: https://doi.org/10.1038/s41467-026-69368-2
Protocol Citation: Chien-Ju Chen 2022. BICCN_DART-FISH. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvmnd45g3p/v1
Manuscript citation:
Palmer CR, Song J, Yang B, Chen C, Diep D, Conklin K, Plongthongkum N, Indralingam HS, Liu CS, Kurtz J, Hu Q, Ransom L, Shahnaee A, Hiniker A, Hodge RD, Keene CD, Lein E, Kharchenko P, Zemke NR, Chun J, Ren B, Zhang K (2026) Single-cell multiomic human brain atlas reveals regulatory drivers of cortical regionality. Nature Communications 17(). doi: 10.1038/s41467-026-69368-2
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: August 23, 2022
Last Modified: August 23, 2022
Protocol  Integer ID: 69012
Keywords: spatial transcriptomic data from human brain section, spatial transcriptomic data, biccn-dart, biccn, fish procedure, protocol documents dart, human brain section, brain
Abstract
This protocol documents DART-FISH procedures used to generate spatial transcriptomic data from human brain section for BICCN.
Materials
Reagents
ABC
materialvendorcatalog number
UltraPure™ DEPC-treated Water 1LThermoFisher Scientific750023
Pierce™ 16% Formaldehyde (w/v), Methanol-freeThermoFisher Scientific28908
PBS - Phosphate-Buffered Saline (10X) pH 7.4, RNase-freeThermoFisher ScientificAM9624
Ethyl alcohol, PureSigma-AldrichE7023
Triton™ X-100 solutionSigma-Aldrich93443
pepsinSigma-Aldrich10108057001
SuperScript™ IV Reverse TranscriptaseThermoFisher Scientific18090010
Advantage® UltraPure dNTP Combination KitClonTech639132
RNase inhibitorEnzymaticsY9240L
Aminoallyl dUTP, 4 mM in TE buffer *UltraPure Grade*, Anaspec IncAnaspec (VWR)AS-83203
BS(PEG)9ThermoFisher Scientific21582
UltraPure™ DNase/RNase-Free Distilled WaterTheroFischer Scientific10977023
Ribonuclease; RNAse H; Conc. 5,000 U/mL; 5,000 U; incl. 10X bufferFisher Scientific50305945
RNase Cocktail™ Enzyme MixInvitrogenAM2288
Ampligase® Enzyme and BufferVWR76081-598
SSC (20X), RNase-freeThermoFisher ScientificAM9763
Formamide (Deionized)ThermoFisher ScientificAM9342
BSA, Molecular Biology GradeNEBB9000S
Phi29 DNA polymerase (10U/uL)ThermoFisher ScientificEP0094
Acryloyl-X, SE in DMSO ThermoFisher ScientificA20770
Acrylamide/Bis-acrylamide, BioReagent, for molecular biology, 37:1 (ratio)Sigma-AldrichA6050-100ML
Ammonium persulfateSigma-AldrichA3678
TEMEDFisher Scientific17919
gel slick solutionLonza50640
TrueBlack lipofuscin autofluorescence quencherBiotium23007
Silicone Isolators JTR20R-2.5 20mm DIA x 2.4 mm Depth 25 x 25mm OD No PSAGrace Bio-Labs664304
Coverslips, Glass, 18mm dia.Ted Pella260369
Azer Scientific cover glass, No. 1.5, 24 x 60 mmNeta ScientificAzer-1152460

Probes
ABC
oligo namevendorsequence
5N_dc10-Cy5_N9IDT/5AmMC12/CCGATAGTCACGATCTGTGGNNNNNNNN*N
dT20_dc7-488IDTCATGGATTCGCGGAGGATCATTTTTTTTTTTTTTTTTTT*T
FISSEQ_ppRCA primerIDTGATATCGGGAAGCTGA*A*G
DARTFISH_anchor_Cy3IDT/5Cy3/CTTCAGCTTCCCGATATCCG
dcProbe7-AF488IDT/5Alex488N/TGATCCTCCGCGAATCCATG
dcProbe10-ATTO647NIDT/5ATTO647NN/CCACAGATCGTGACTATCGG
dcProbe0-AF488IDT/5Alex488N/TGTATCGCGCTCGATTGGCA
dcProbe0-Cy3IDT/5Cy3/CGTATCGGTAGTCGCAACGC
dcProbe0-ATTO647NIDT/5ATTO647NN/ACGCTACGGAGTACGCCACT
dcProbe1-AF488IDT/5Alex488N/TCTTGCGTGCGATACGGAGT
dcProbe1-Cy3IDT/5Cy3/AACGGTATTCGGTCGTCATC
dcProbe1-ATTO647NIDT/5ATTO647NN/CTGGTTCGGGCGTACCTAAC
dcProbe2-AF488IDT/5Alex488N/AGAACTTGCGCGGATACACG
dcProbe2-Cy3IDT/5Cy3/CTACTTCGTCGCGTCAGACC
dcProbe2-ATTO647NIDTGACGAACGGTCGAGATTTAC/3ATTO647NN/
dcProbe3-AF488IDT/5Alex488N/GAATTGTCCGCGCTCTACGA
dcProbe3-Cy3_2IDT/5Cy3/TCGTACTTCGACGGCACTCA
dcProbe3-ATTO647NIDT/5ATTO647NN/AACTGCGACCGTCGGCTTAC
dcProbe4-AF488IDT/5Alex488N/CGGAATACGTCGTTGACTGC
dcProbe4-Cy3IDT/5Cy3/TACCATTCGCGTGCGATTCC
dcProbe4-ATTO647N_2IDT/5ATTO647NN/ACTCTACCGGCAATCGCGTC
dcProbe5-AF488IDT/5Alex488N/GAGTGTCGCGCAACTTAGCG
dcProbe5-Cy3IDT/5Cy3/ACGTCTGCGTACCGGCTTAG
dcProbe5-ATTO647NIDT/5ATTO647NN/CATGCGATTAACCGCGACTG
dcProbe6-AF488_2IDT/5Alex488N/CTTGCGGCGACAGTCGAACA
dcProbe6-Cy3IDT/5Cy3/TCGTAACCCGTGCGAAGTGC
dcProbe6-ATTO647NIDT/5ATTO647NN/CTCTCGTAGCGTGCGATGAG
dcProbe7-AF488_2IDT/5Alex488N/TTAGGTCGCCTACCGACTGC
dcProbe7-Cy3IDT/5Cy3/GCCACATCGACTCGGTCTAT
dcProbe7-ATTO647NIDTGCTCAGCCGGACGAGTAGAT/3ATTO647NN/

preparation
10m
Make sure that the padlock probes have 5' phosphate. In case they do not, run T4 PNK reaction and clean up the product using Zymo ssDNA/RNA clean up columns.
Wash, dry and UV the silicone isolators. UV the EasyDip jars. Move away unused stuff from the bench and clean and undust the working area. Set the HybEZ oven 37C.
permeabilization
Prepare 75ml of 4% formaldehyde in 1x PBS. Requires two vials of 16% formaldehyde.
AB
componentvolume (mL)
DEPC-water52
16% PFA20
10X PBS8

Take the sections that are on 25mm*60mm coverslips out of -80C on dry ice, insert them into the slide holder, submerge the slide holder in the staining jar containing 4% PFA in PBS. Fix for 60mins at 4C.
01:00:00 4C
1h
Remove the DEPC-PSBT jar and the 4% PFA jar containing the samples from 4C. Insert the sample holder into the cold 1x PBST jar. Incubate for 3 minutes. Then insert the sample holder in the room temperature 1x PBST jar. Incubate for 3 minutes.
00:03:00 in 4C DEPC-PBST with occasional agitation
00:03:00 in RT DEPC-PBST with occasional agitation
6m
dehydration and mounting
20m
Prepare jars of 50%, 70% and two 100% ethanol. Dehydrate Section with
00:05:00 50% EtOH at room temperature
00:05:00 70% EtOH at room temperature
00:05:00 100% EtOH at room temperature
00:05:00 100% EtOH at room temperature
20m
Take the coverslips out of the sample holder.
00:05:00 Air drying at room temperature.
In the meantime, put 20mm diameter Press-To-Seal silicone isolators on a kipwipe on a flat surface. Carefully put the coverslips on silicone isolators, with the tissue sample in the hole. Gently press on the back of the coverslip to seal completely. You may want to stack a 20mm diameter isolator on top of the already mounted 20mm isolator to increase the volume.
5m
permeabilization
10m
Permeabilize sample with 0.25% Triton X-100 in DEPC-1X PBS for 00:10:00 at Room temperature
875 µL DEPC-H2O 100 µL 10x PBS 25 µL 10% Triton-X 100

AB
componentx1 volume (uL)
DEPC-water875
10X PBS100
10% Triton X-10025

10m
Wash thrice with DEPC-1X PBS and DEPC-Water
1 mL DEPC-1X PBS quick wash
1 mL DEPC-1X PBS quick wash
1 mL DEPC-water quick wash
pepsin digestion
10m
Digest with 0.01% Pepsin in 0.1N HCl. Pre-warm the pepsin to 37 °C for at least 5 minutes before use.
100 µL 0.01% Pepsin in 0.1N HCl for00:01:30 at 37 °C
AB
componentx1 volume (uL)
1% pepsin1
0.1N HCl99

1m 30s
Wash twice with DEPC-1X PBS
1 mL DEPC-1X PBS quick wash
1 mL DEPC-1X PBS quick wash
reverse transcription
15m
Prepare Reverse Transcription Mix on Ice.
AB
componentx1 volume (uL)
DEPC-water90.75
5X SSIV buffer30
10mM dNTP3.75
100uM N5_dc10-Cy5_N93.75
100uM dT20_dc7-4883.75
0.1M DTT7.5
4mM aminoallyl-dUTP1.5
RNase inhibitor (40U/uL)1.5
SuperScript IV Reverse Transcriptase7.5
total150
Incubate human brain sections with the Reverse Transcription Mix
150 µL Reverse Transcription Mix for 00:10:00 at 4 °C then Overnight at 37 °C
15m
Wash with 1X PBS twice.
1 mL 1X PBS quick wash
1 mL 1X PBS quick wash
cDNA crosslinking
1h 30m
Add the crosslinking mix to crosslink cDNAs with BS(PEG)9. Prepare 5mM BS(PEG)9 in PBS.

AB
componentx1 volume (uL)
250mM BS(PEG)910
10X PBS50
ultrapure water440
Crosslink cDNA with BS(PEG)9
500 µL 5 mM BS(PEG)9 in PBS for 01:00:00 at Room temperature

Wash with PBS twice
1 mL 1X PBS quick wash
1 mL 1X PBS quick wash

Quench unreacted crosslinker with 1M Tris, pH 8.0
1 mL 1M Tris pH8.0 for 00:30:00 at Room temperature

Wash with PBS twice
1 mL 1X PBS quick wash
1 mL 1X PBS quick wash
1h 30m
RNase digestion
1h
Prepare RNase Digestion Mix

AB
componentx1 volume (uL)
ultrapure water168
10X RNase H buffer20
RNase H (5U/uL)10
RNase Cocktail2

Add 200 µL RNase Digestion Mix
Incubate at 37 °C for 01:00:00 . Cover the silicone wells.
1h
Wash samples with 1X PBS twice.
1 mL 1X PBS quick wash
1 mL 1X PBS quick wash
padlock probe hybridization
33m
Prepare the 484-gene-Hybridization Mix according to the table below. Preheat the probe-water mix to 85 °C for 00:03:00 and immediately move them to a cold block or on ice. Then complete the Hybridization Mix.

484-gene-Hybridization Mix
AB
componentx1 volume (uL)
ultrapure water98.2
10X Ampligase buffer15
HB_Feb2022 probes ( 27.2 ng/uL)25.3
100nM PLP1 oligos1.5
Ampligase (5U/uL)10



add 150 µL 484-gene-Hybridization mix to human section

3m
Incubate samples in the Hybridization Mix at 37 °C for 00:30:00 , then at 55 °C Overnight .
For the overnight incubation, first set the Ez hyb oven to 60 °C and then change it to 55 °C as you put the samples in. Cover the sample well.
30m
Wash samples with 1x PBS twice.
1 mL 1X PBS quick wash
1 mL 1X PBS quick wash
RCA
6h
Prepare RCA Primer Mix
AB
componentx1 volume (uL)
ultrapure water119
20X SSC20
formamide60
100 uM FISSEQ_ppRCA primer 1
add 200 µL RCA Primer Mix to each sample and incubate at 37 °C for 01:00:00

1h
Wash samples with 2xSSC.
1 mL 2X SSC quick wash
1 mL 2X SSC quick wash
Prepare RCA Enzyme Mix on ice.
AB
componentx1 volume (uL)
ultrapure water119.25
10X Phi29 polymerase buffer15
10mM dNTP3.75
4mM aminoallyl-dUTP1.5
NEB BSA (20mg/mL)7.5
ThermoFisher Phi29 polymerase3
total150
Add 150 µL RCA Enzyme Mix to each sample
Incubate samples in RCA Enzyme Mix at 30 °C for 05:00:00

5h
Wash samples with 1x PBS twice.
1 mL 1X PBS quick wash
1 mL 1X PBS quick wash
rolony crosslinking
1h 30m
Crosslink with Acryloyl-X Mix and embed the sample in gel.

Prepare Acryloyl-X Mix
AB
Componentx1 volume (uL)
10X PBS50
10mg/mL Acryloyl-X, SE in DMSO10
ultrapure water440
add 500 µL Acryloyl-X Mix to the sample and incubate for 00:30:00 at Room temperature

quick wash with 1xPBS.
1 mL 1X PBS quick wash

Incubate the sample with 300 µL Acrylamide Solution for 00:30:00 at Room temperature .
Prepare Acrylamide Solution.
AB
Componentx1 volume (uL)
10X PBS50
40% Acrylamide/Bis (37:1)50
ultrapure water400
Aspirate the Acrylamide Solution. Do not wash the samples!!!

Prepare Polymerization Mix.
Prepare 5% TEMED: 5 µL TEMED in 95 µL ultrapure water
Prepare 4% APS: 10 mg Ammonium Persulfate in 250 µL ultrapure water
RNaseZap and UV 18mm coverslips and treat them with Gel-Slick.

AB
componentx1 volume (uL)
Acrylamide solution138
4% APS6
5% TEMED6

Add 30 µL Polymerization Mix to the sample and cover with Gel-Slick-treated coverslip for 00:30:00 at Room temperature in an Argon tank.

wash with 1x PBST for 3min twice
1 mL 1X PBST for 00:03:00
1 mL 1X PBST for 00:03:00

Carefully remove the Gel-Slick-treated coverslip on the samples

wash with 1xPBST for 3min twice
1 mL 1X PBST for 00:03:00
1 mL 1X PBST for 00:03:00
1h 42m
imaging
11m
stain the sample with Probe Hybridization Mix with decoding probes (Take dcProbe0_AF488, dcProbe0_Cy3, dcProbe0_ATTO647N probes as an example).

Prepare Probe Hybridization Mix
AB
componentx1 volume (uL)
100 uM dcProbe0_AF4881
100 uM dcProbe0_Cy31
100 uM dcProbe0_ATTO6471
100% formamide60
20X SSC20
ultrapure water117

add 200 µL Probe Hybridization Mix to each sample. Incubate for 00:08:00 at Room temperature

8m
Wash with10% formamide in 2X SSC twice. Then, image.
1 mL 10% formamide in 2X SSC for 00:03:00
1 mL 10% formamide in 2X SSC for 00:03:00

6m
Strip with 1 mL 80% formamide in 2X SSC for 00:05:00 .
Wash with 1 mL 2X SSC buffer once.
Repeat the decoding imaging with the next decoding probes (dcProbe1, dcProbe2, dcProbe3, dcProbe4, dcProbe5, dcProbe6, and dcProbe7).

5m
After images of samples stained with dcProbe0, 1, 2, 3, 4, 5, 6, 7 were taken, take the nuclei staining images with Draq5 staining.

add 500 µL Draq5 solution to the sample. Incubate for 00:05:00 at Room temperature

wash the sample with 1x PBS twice. Then, image.
1 mL 1X PBS for 00:02:00
1 mL 1X PBS for 00:02:00

9m