TITLE: Multiplexed, single-cell profiling of histone modifications with SCEPTRE
AUTHORS: Marcus  A Woodworth, Hao Yuan Kueh, Joshua C. Vaughan

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Genome architecture and activity are tightly regulated during an organism’s development and function. Histone modifications are thought to contribute to this regulation by acting in combination to specify the activity states of individual genes. However, most methods used to profile these modifications either require a large number of cells or only target an individual histone modification at a time. This protocol uses the method Single Cell Evaluation of Post-TRanslational Epigenetic Encoding (SCEPTRE) to quantify immunolabeled H3K4me3 and H3K27me3 histone modifications at specific non-repetitive genomic loci in single cells using Expansion Microscopy. The protocol can be adapted to target alternative histone modifications or protein assemblies in other cell lines and for other genomic regions of interest.</div><div class = "text-block">Reference:</div><div class = "text-block">Woodworth,M.A., Ng,K.K.H., Halpern,A.R., Pease,N.A., Nguyen,P.H.B., Kueh,H.Y. and Vaughan,J.C. (2021) Multiplexed single-cell profiling of chromatin states at genomic loci by expansion microscopy. Nucleic Acids Research, 10.1093/nar/gkab423.</div></div>

[STEPS]
?. [Cell Culture and Fixation]
Wash three times with  of 1× PBS.
250 µl
?. [Immunofluorescence]
Wash cells three times with  of 1× PBS.
250 µl
?. [Immunofluorescence]
Incubate in   of blocking solution for   at  .
250 µl
0 Room temperature
?. [Immunofluorescence]
Incubate in  of primary solution    at  .
250 µl
[~18 hours]
4 °C
?. [Immunofluorescence]
Wash the sample with   of blocking solution three times,  each time.
250 µl
?. [Immunofluorescence]
Incubate in  of secondary solution for   at  .
250 µl
0 Room temperature
?. [Immunofluorescence]
Wash once for  with  of blocking solution.
250 µl
?. [Immunofluorescence]
Wash three times with  of 1× PBS.
250 µl
?. [Immunofluorescence]
Post-fix in   of freshly made 4% PFA in 1× PBS for  .
250 µl
?. [Immunofluorescence]
Wash three times with  of 1× PBS.
250 µl
?. [Gelation, Digestion and Expansion.]
Treat immunolabeled cells with   of freshly prepared   MA-NHS in 1× PBS for  .
250 µl
?. [Gelation, Digestion and Expansion.]
Wash samples three times with  of 1× PBS.
250 µl
?. [Gelation, Digestion and Expansion.]
Incubate in  of ExM monomer solution for .
250 µl
?. [Gelation, Digestion and Expansion.]
Remove coverslip with cell sample from 24-well plate and place, cells facing down, on to  of ExM monomer solution supplemented with 0.15% (w/v) APS and 0.2% (w/w) TEMED.
50 µl
?. [Gelation, Digestion and Expansion.]
Gently remove the cell-embedded hydrogel from the 12 mm coverslip.
?. [Gelation, Digestion and Expansion.]
Incubate cell-embedded hydrogel in  of digestion solution at .
1 mL
37 °C
?. [Gelation, Digestion and Expansion.]
Wash and expand by placing the digested sample in  of deionized water, replacing the water every 15-20 minutes for at least three times or until gel sample is ~4× the original size.
50 mL
?. [DNA Fluorescence in situ hybridization]
Slice a small (~3.5 mm × 3 mm × 2 mm, equivalent to  ) piece of the hydrogel sample.
20 µl
?. [DNA Fluorescence in situ hybridization]
Remove excess buffer and incubate gel slice in  of pre-heated hybridization buffer for  at .
500 µl
60 °C
?. [DNA Fluorescence in situ hybridization]
Remove the excess buffer from the gel slice and add  of pre-heated hybridization mixture to the gel slice.
50 µl
?. [DNA Fluorescence in situ hybridization]
Denature sample at  for .
90 °C
?. [DNA Fluorescence in situ hybridization]
Remove excess buffer and wash gel slice by incubating in  of preheated wash buffer for  at .
500 µl
60 °C
?. [DNA Fluorescence in situ hybridization]
Remove excess buffer and wash gel slice by incubating in  of wash buffer for  at .
500 µl
37 Room temperature
?. [Expansion, imaging and analysis]
Remove excess buffer, then expand gel slice to ~4× by adding and replacing  of deionized water, twice every  at  .
500 µl
4 °C
?. [Cell Culture and Fixation]
Grow h-TERT RPE1 with cell culture media to ~80% confluency.
?. [Cell Culture and Fixation]
Briefy rinse cells with   of 1× PBS.
250 µl
?. [Immunofluorescence]
Place fixed RPE1 cells in   permeabilization buffer for  .
250 µl
?. [Cell Culture and Fixation]
Trypsinize cells with 0.25% trypsin-EDTA and seed at ~75,000 cells per well on top of round coverslips (no. 1.5, ~12 mm diameter) placed within 24-well culture plates.
?. [Cell Culture and Fixation]
Grow cells at  .
[in 4% carbon dioxide humidified enviorment]
?. [Cell Culture and Fixation]
Fix cells with  of cold EtOH:MeOH (1:1) for  at  .
250 µl
-20 °C
?. [Gelation, Digestion and Expansion.]
Gel sample at   for  in a sealed container backfilled with nitrogen gas.
0 Room temperature
?. [DNA Fluorescence in situ hybridization]
Incubate gel slice in   of hybridization buffer for  at .
1 mL
0 Room temperature
?. [DNA Fluorescence in situ hybridization]
Make   of hybridization mixture and pre-heat to  for .
50 µl
90 °C
?. [DNA Fluorescence in situ hybridization]
Hybridize probes to sample  at  .
42 °C
?. [DNA Fluorescence in situ hybridization]
Remove excess buffer and wash gel slice by incubating in  of preheated wash buffer for  at .
500 µl
37 °C
?. [DNA Fluorescence in situ hybridization]
Remove excess buffer and wash gel slice by incubating in  of  one tenth diluted wash buffer for .
500 µl
on ice
?. [Cell Culture and Fixation]
Store fixed cells in  of 1× PBS azide (1× PBS with 3 mM sodium azide) at .
250 µl
4 °C
?. [Immunofluorescence]
Store immunofluorescently labeled cells in  of 1× PBS with 3mM sodium azide at .
250 µl
4 °C
?. [Gelation, Digestion and Expansion.]
Store hydrogel sample at  in 2× SSC buffer.
4 °C
?. [DNA Fluorescence in situ hybridization]
Store samples at  .
4 °C
?. [Expansion, imaging and analysis]
Place the expanded gel slice on a poli-lysine treated coverslip, cell-side facing down.
?. [Expansion, imaging and analysis]
Image sample with a confocal microscope to obtain 3D image stacks of the nuclei of each cell.
?. [Expansion, imaging and analysis]
Analyze fluorescence intensity of each histone modification at the individual loci to determine the activity state of each locus.