TITLE: 5: User-friendly protocol: SABER RNA FISH in cells
AUTHORS: Jocelyn Y. Kishi, Sylvain W. Lapan, Brian J Beliveau, Emma R. West, Allen Zhu, Hiroshi M. Sasaki, Sinem Saka, Yu Wang, Constance L Cepko, Peng Yin

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes the SABER RNA FISH in cells.</div></div>

[STEPS]
?. [Preparing the slide]
Seed 8-well Ibidi chamber slides (Cat #80827) with cells and grow in tissue culture incubator (typically  with ) to desired confluency.
37 °C
[CO2]
Notes: You may need to adjust the deposition protocol (e.g. what concentration of cells, how long to let them grow on the slide) in order to achieve the desired confluency of your cell type for imaging.Some cell types will not adhere well through the fixation protocol, so the chamber should be quickly imaged in Brightfield after fixation to ensure the proper density of cells.If this is your first time doing this protocol, we recommend using some of the wells for controls and a couple probe length conditions. For example, you might seed 4 wells and use one as a no probe control well (which receives all other treatments) where you should not see signal, and then 3 different concatemer length conditions. Or if you have 3 targets, you might do 3 single-color wells each receiving one of the concatemer species, and then one multi-color well. We have found that some microscope stages are not amenable to imaging the leftmost and rightmost wells in the chamber, so this is a good thing to check before you plan on using all of the wells. Depending on the type of fluid, the minimum volume for each well is around  - . We recommend using   for washes. If you are using a different type of chamber with smaller volume, we recommend performing several more washes at each step to ensure complete removal of previous elements. The easiest method of aspiration is with an unfiltered pipette tip attached to a vacuum line. We recommend cleaning the line with ethanol and changing the tip a several times throughout the experiment. Good lab technique for avoiding RNase contamination and use of RNase-free water is important throughout the protocol.
?. [Fixation]
At , rinse cells in  and then immediately fix in  for .
37 Room temperature
[PBS]
[ paraformaldehyde]
?. [Fixation]
Rinse again in  and store at .
[PBS]
4 °C
We stored chambers for DNA FISH up to a couple of weeks before use, but for RNA integrity we recommend waiting no longer than a few days.
?. [Hybridization]
Wash in  for  at .
[PBS]
4 Room temperature
?. [Hybridization]
Permeabilize in  +  for  at .
[PBS]
[Triton X-100]
4 Room temperature
?. [Hybridization]
Wash in  for  at .
[PBSTw]
4 Room temperature
?. [Hybridization]
Wash in  for  at .
[SSCT]
4 Room temperature
?. [Hybridization]
Add hybridization solution and denature at  for .
60 °C
?. [Hybridization]
Return temperature to  for at least  (typically  is easiest).
42 °C
Note: if the probes are retrieved using the Oligominer33 pipeline as described or from the Wu lab database, then   hyb temperature should be sufficient for RNA FISH. If you are using custom designed and especially shorter probes, you will need to check their melting temperatures in   +  . We suggest setting your oven temperature to a couple degrees lower than those melting temperatures (See protocol 3). We have found that thermocyclers especially with heated lids versus ovens set to the same temperature can actually be up to a few degrees higher at the sample plane. This can cause a big difference in yield because we are operating close to the melting temperatures, so if you feel that your signal is too low you can try reducing the temperature of the hyb a bit. Alternatively, if there seems to be a lot of off target binding of probes, you can try increasing a bit.
?. [Hybridization]
Add  pre-warmed  to wells and aspirate.
200 µl
[SSCT]
Note: For pre-warming  , we suggest you first make enough for all washes and then aliquot the amount needed for the hot washes into 2 mL tubes. Put these tubes on a heated tube rack set to   and allow them to heat for at least  - . Remove only the amount of tubes you need at each wash step, so that the rest can stay hot.
?. [Hybridization]
Wash 4× in pre-warmed  at .
[SSCT]
60 °C
?. [Hybridization]
Wash  in pre-warmed  at . (1/4)
[SSCT]
60 °C
?. [Hybridization]
Wash 2× in  at .
[SSCT]
60 Room temperature
?. [Hybridization]
Wash  in  at . (1/2)
[SSCT]
60 Room temperature
?. [Hybridization]
If you are going directly to the fluorescent protocol, rinse in  for  and transfer to fresh  (at).
[PBS]
[PBS]
60 Room temperature
?. [Hybridization]
Pause point: sample can be stored at  to several days.
4 °C
?. [Hybridization]
Wash  in pre-warmed  at . (2/4)
[SSCT]
60 °C
?. [Hybridization]
Wash  in pre-warmed  at . (3/4)
[SSCT]
60 °C
?. [Hybridization]
Wash  in pre-warmed  at . (4/4)
[SSCT]
60 °C
?. [Hybridization]
Wash  in  at . (2/2)
[SSCT]
60 Room temperature
?. [Branching]
Wash in  for  at .
[SSCT]
65 Room temperature
?. [Branching]
Add branch hybridization solution and hold at  for at least .
37 °C
Note: Branching is performed similarly to primary probe incubation, but in a lower formamide solution. Branches are applied after primary probe washes are complete, and before fluorescent detection. For cells, branches are extended to length of ~250-450 nt and incubated for at least 30 minutes in  . Hyb temperature must be adjusted depending on the branches being used. We recommend using a temperature at least 1 degree lower than the lowest melting temperature of your branch sequences (see Fig. S2, protocol 3). Note that formamide concentration can be adjusted instead of oven/thermocycler temperature.
?. [Branching]
Add  pre-warmed  to wells and aspirate.
200 µl
[SSCT]
?. [Branching]
Wash 4× in pre-warmed  at .
[SSCT]
60 °C
?. [Branching]
Wash  in pre-warmed  at . (1/4)
[SSCT]
60 °C
?. [Branching]
Wash  in pre-warmed  at . (2/4)
[SSCT]
60 °C
?. [Branching]
Wash  in pre-warmed  at . (3/4)
[SSCT]
60 °C
?. [Branching]
Wash  in pre-warmed  at . (4/4)
[SSCT]
60 °C
?. [Branching]
Wash 2× in  at .
[SSCT]
60 Room temperature
?. [Branching]
Wash  in  at . (1/2)
[SSCT]
60 Room temperature
?. [Branching]
Wash  in  at . (2/2)
[SSCT]
60 Room temperature
?. [Branching]
Rinse in  for .
[PBS]
?. [Branching]
Transfer to fresh  (at ).
[PBS]
60 Room temperature
?. [Fluorescent detection]
Rinse once in  at .
[PBS]
60 Room temperature
?. [Fluorescent detection]
Add fluor hybridization solution and hold at  for .
37 °C
Notes: We recommend starting with the 1 hour hybridization with    if this is your first time running the protocol. Alternatively, we still see strong signal if the hybridization time is reduced to   (at   e.g. for Fig. 6 in the Nature Methods article). Side-by-side testing with two probes in tissue indicate  is also sufficient (see Fig. S3, protocol 6).
?. [Fluorescent detection]
Wash for  with pre-warmed  at .
[PBS]
37 °C
Note: For pre-warming  , we suggest you first make enough for all washes and then aliquot the amount needed for the warming washes into a separate Falcon tube and place into an oven or warm room at   -  .
?. [Fluorescent detection]
Wash 2× in pre-warmed .
[PBS]
?. [Fluorescent detection]
Wash  in pre-warmed . (1/2)
[PBS]
?. [Fluorescent detection]
Wash  in pre-warmed . (2/2)
[PBS]
?. [Fluorescent detection]
Rinse once in  at .
[PBS]
45 Room temperature
?. [Fluorescent detection]
Load SlowFade + DAPI (Thermo Fisher S36939) mountant to completely cover cells.
Note: You can also image samples directly in  , in which case if you may need to do a short DAPI stain if you want to see that channel.
?. [Fluorescent detection]
Image.
?. [Fluorescent detection]
Pause point: Samples can be stored at  before imaging for a few days, although we recommend doing the fluorescent hyb closer to your imaging session time (day of) where possible.
4 °C
?. [Serial detection (complete before repeating fluorescent detection)]
Wash 2× in PBS at  to remove mountant.
4 Room temperature
?. [Serial detection (complete before repeating fluorescent detection)]
Wash  in PBS at  to remove mountant. (1/2)
4 Room temperature
?. [Serial detection (complete before repeating fluorescent detection)]
Wash  in PBS at  to remove mountant. (2/2)
4 Room temperature
?. [Serial detection (complete before repeating fluorescent detection)]
Add displacement buffer and incubate for  at .
4 Room temperature
Notes: the signal should be stripped almost immediately with the displacement buffer, but we recommend letting it sit for at least  -  to ensure complete removal. If you are using a smaller volume chamber or well, you may need to add fresh displacement buffer extra times in order to completely strip fluorescent signal. If you are doing the exchange in place on a microscope, you can image the same area before and after adding the displacement buffer to visualize the drop in signal. If you are using DAPI, the displacement buffer will probably remove most of this signal. You will likely need to replace after each exchange step and before imaging, either by including it in the mountant or staining separately (see above).