Jan 20, 2025
Multi-round smHCR-FISH for archaea
- Heng Xu1,
- Fengping Wang1
- 1Supervisor
Protocol Citation: Heng Xu, Fengping Wang 2025. Multi-round smHCR-FISH for archaea. protocols.io https://dx.doi.org/10.17504/protocols.io.261ger9qyl47/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: January 18, 2025
Last Modified: January 20, 2025
Protocol Integer ID: 118654
Keywords: smHCR-FISH
Funders Acknowledgements:
the National Natural Science Foundation of China
Grant ID: 41921006
the National Natural Science Foundation of China
Grant ID: 92251303
the National Natural Science Foundation of China
Grant ID: 42230401
the National Natural Science Foundation of China
Grant ID: 11774225
the National Natural Science Foundation of China
Grant ID: 42406089
the National Key R&D Program of China
Grant ID: 2021YFA0910702
the Natural Science Foundation of Shanghai
Grant ID: 22ZR1434000
the 2030 program of Shanghai Jiao Tong University
Grant ID: WH510244001
Abstract
Understanding archaeal gene regulation is technically challenging due to the difficulty of measuring gene expression in individual archaeal cells. Here, we develop a multi-round smHCR-FISH method to quantify the transcription of multiple genes in single cells of a methanogenic archaeon.
Materials
CRITICAL Use RNase- and DNase-free materials whenever possible.
Diethylpyrocarbonate (DEPC)-treated water, Rnase free (Ambion/Invitrogen, cat. no. 4387937)
10*PBS, Rnase free (Ambion/Invitrogen, cat. no.AM9624)
Formamide, deionized, nuclease free (Ambion/Invitrogen, cat. no.AM9342)
Sodium Chloride (Fisher Scientific, cat. no. BP358-1)
RNase Zap (Sigma, cat. no.R2020-250ml)
8% paraformaldehyde (EMS, cat. no.157-8, 10◊10ml)
Aqua-poly/mount (Polysciences, cat. no. 18606)
Ethanol, for molecular biology, absolute 500ml (Fisher chemical, cat. no. BP2818-500)
Tween 20 (Sigma, cat. no. P9416-50ml)
0.5M Na2HPO4 solution (Sigma, cat. no. 94046-100ml)
0.5M EDTA, PH 8.0, RNase free (Fisher Scientific, cat. no. AM9260G)
1M TRIS-HCl (ShengGong, cat. no.B548138-0500)
Dextran sulfate sodium salt (Sigma, cat. no. D8906)
20*SSC, RNase free (Invitrogen,AM9770)
Hoechst 33342 (Invitrogen, cat. no. H3570)
50*Denhardt (Fisher Scientific, cat. no. 750018)
Poly-L-Lysine (Sigma, cat. No. P4707)
HCR-FISH DNA probes with fluorescence dye labeled.
DNase I (TURBO DNase, AM2238)
popoTM-1 (Invitrogen, P3580)
4-chamber glass-bottom dish (Cellvis,D35C4-20-1-N)
Zeiss LSM 880 laser scanning confocal microscope equipped with an Airyscan detector and a plan-Apochromat 63×/1.4 NA oil-immersion objective
Cell fixation:
Cell fixation:
For smHCR-FISH experiments, 3 mL of sampled culture was fixed with 1 mL of 8% (v/v) paraformaldehyde for
12 h at 4 °C, then washed twice with 1× PBS (10 min each), and stored in a 1:1 (v/v) mixture of ethanol and 1× PBS at -20 °C.
Multi-round smHCR-FISH and imaging
Multi-round smHCR-FISH and imaging
A 4-chamber glass-bottom dish (Cellvis, D35C4-20-1-N) was bottom-coated with poly-L-Lysine (Sigma, P4707). Fixed cells were settled onto the bottom of a well for two hours at room temperature. The dish was then mounted in the incubation system of a Zeiss LSM 880 laser scanning confocal microscope for multi-round smHCR-FISH.
For each round of the experiment, cells in each well were incubated in 40 mL of hybridization buffer (20 mM Tris-HCl, 25% (w/v) formamide, 250 mM EDTA, 2% (v/v) 50× Denhardt's solution, and 0.1% (v/v) Tween 20) with primary probes (0.1 mM for each gene) at 46 °C for 4 h.
Following hybridization, cells were washed three times (20 min each) at 48 °C in 500 mL of wash buffer (159 mM
NaCl, 20 mM Tris-HCl, 0.1% Tween 20).
For HCR amplification, each amplifier probe was separately dissolved in amplification buffer (5× SSC, 10% dextran sulfate, 0.1% Tween 20, 150 nM probe), heated to 95 °C for 2 min, and cooled to room temperature for 30 min to open the hairpin structure. Cells were incubated with a 20-mL-mixture of all amplifier probes at 35 °C in a humid atmosphere for 1 h.
After HCR amplification, cells were washed three times (10 min each) in 500 mL 4× SSC and stained with 1:10000 diluted Hoechst 33342 (Invitrogen, H3570) for 10 minutes. Following three additional rinses in 4× SSC, cells were covered by a mixture of Aqua-poly/Mount (Polysciences, 18606) and 4× SSC (~3:10 (v/v)) for imaging.
Image acquisition was performed using a Zeiss LSM 880 laser scanning confocal microscope equipped with an Airyscan detector and a plan-Apochromat 63×/1.4 NA oil-immersion objective.
After imaging, HCR-FISH probes in the cell were removed using DNase I (TURBO‱ DNase, AM2238). Briefly, cells in each well were washed in 50 mL of 1× DNase buffer for 5 min and incubated with 3 mL DNase I (2 Units/mL) in 1× DNase buffer at 37 ℃ for 2 hours.
Following enzyme digestion, cells were washed three times (10 min each) in wash buffer at 37 ℃ and fixed in 2% PFA for 15 min at room temperature to inactivate DNase I.
After three more washes (10 mineach) in 4× SSC, samples were ready for the next round of the experiment. From
the second round on, since cellular DNA had been degraded in DNase I treatment, cellular RNA, instead of DNA, was stained for cell identification using popoTM-1(Invitrogen,P3580).
Protocol references
1. Yamaguchi, T. et al. Rapid and sensitive identification of marine bacteria by an improved in situ DNA hybridization chain reaction (quickHCR-FISH). Syst. Appl. Microbiol. 38, 400-405 (2015).
2. Choi, H.M.T. et al. Mapping a multiplexed zoo of mRNA expression. Development 143, 3632-3637 (2016).
3. Morono, Y., Kubota, K., Tsukagoshi, D. & Terada, T. EDTA-FISH: A Simple and Effective Approach to Reduce Non-specific Adsorption of Probes in Fluorescence in situ Hybridization (FISH) for Environmental Samples. Microbes Environ. 35, ME20062 (2020).
4. Shah, S., Lubeck, E., Zhou, W. & Cai, L. In situ transcription profiling of single cells reveals spatial organization of cells in the mouse hippocampus. Neuron 92, 342-357 (2016).
Acknowledgements
This work was supported by the National Natural Science Foundation of China (grant no. 41921006, 92251303, 42230401, 11774225, 42406089), the National Key R&D Program of China (grant no. 2021YFA0910702), and the
Natural Science Foundation of Shanghai (grant no. 22ZR1434000). This is also a contribution to the 2030 program of Shanghai Jiao Tong University (WH510244001) and the International Center for Deep Life Investigation (IC-DLI). We
gratefully acknowledge the Student Innovation Center at Shanghai Jiao Tong University.