TITLE: In situ sequencing for RNA analysis in tissue sections
AUTHORS: Chika Yokota, Daniel Gyllborg, Mats Nilsson

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">In situ sequencing method for parallel targeted analysis of short RNA fragments in morphologically preserved tissue. This protocol can be used to detect RNA molecules at the single cell level to aid in the identification of cell types according to their gene expression. The technique uses padlock probes to target desired genes of interest and rolling circle amplification to amplify signal for a high throughput methodolgy of spatial transcriptomics. With the use of barcode sequencing, identification of numerous genes is possible through multiplexing.</div><div class = "text-block">Version 2 Update: Only minor mistakes were corrected for version 2 of this protocol, no major changes to protocol were done. Some references were also updated.</div></div>

[STEPS]
?. [Tissue section sample preparation]
Fresh frozen tissue samples embedded in OCT compound and stored at -80°C. Tissue is cryosectioned at 10 μm and collected on Superfrost Slides and can be stored at -80°C until fixation and start of procedure.(see Note 9)
?. [Tissue section sample preparation]
Slides are taken from -80°C and left at room temperature (RT) for 3 min to air dry.Fixation performed with freshly prepared 3% (w/v) paraformaldehyde in DEPC-PBS for 5 min at RT.PFA solution is applied directly on top of the section.(see Note 10)
[Thawing]
Safety precautions: paraformaldehyde
[Fixation]
?. [Tissue section sample preparation]
Sections rinsed with DEPC-PBS two times (x2).
?. [Tissue section sample preparation]
The tissue is permeabilized with 0.1 M HCl (in H2O) at RT for 5 min.Glass slide is dipped in a ~20 ml solution of 0.1 M HCl.
Safety precautions: HCl
[HCl treatment]
?. [Tissue section sample preparation]
After permeabilization, slides are washed in DEPC-PBS for 1 min at RT.Rinse slide 2x more times with DEPC-PBS
[PBS Wash]
?. [Tissue section sample preparation]
Slides are dehydrated in an ethanol (EtOH) series.
70% EtOH, RT, 1 min

100% EtOH, RT, 1 min
[70% EtOH]
[100% EtOH]
?. [Tissue section sample preparation]
Slide sections are air dried. When dry, SecureSeal hybridization chambers mounted over tissue. Use a chamber size that covers your desired tissue.
?. [Tissue section sample preparation]
Sections are rehydrated with DEPC-PBS-T and then once with DEPC-PBS.(see Note 11)
?. [In situ reverse transcription]
Reagents for reverse transcription were combined as in the table below.Note: All following tables in this protocol have been calculated to a final volume of 50 µl and should be adjusted accordingly.
?. [In situ reverse transcription]
ABCD1Reagents[Stock][Final]1x (µl)2DEPC-H2O34.753Reverse Transcriptase Buffer10x1x54dNTPs25 mM500 µM15BSA20 µg/µl0.2 µg/µl0.56Random Decamers100 µM5 µM2.57Riboprotect RNase Inhibitor40 U/µl1 U/μl1.258TranscriptME Reverse Transcriptase200 U/µl20 U/μl59Total&nbsp;&nbsp;50
ABCD1Reagents[Stock][Final]1x (µl)2DEPC-H2O34.753Reverse Transcriptase Buffer10x1x54dNTPs25 mM500 µM15BSA20 µg/µl0.2 µg/µl0.56Random Decamers100 µM5 µM2.57Riboprotect RNase Inhibitor40 U/µl1 U/μl1.258TranscriptME Reverse Transcriptase200 U/µl20 U/μl59Total&nbsp;&nbsp;50
?. [In situ reverse transcription]
DEPC-PBS removed and combined reagents added to seal chamber.Chamber is sealed and slide placed in a humidity chamber.Slide and chamber is incubated from 6 hours to overnight at 37°C.
[Incubator]
?. [Barcode padlock probe hybridization and ligation]
Reverse transcription reagents are removed and postfixation performed with 3% (w/v) paraformaldehyde in DEPC-PBS for 30 min at RT.
[PFA fixation]
?. [Barcode padlock probe hybridization and ligation]
Wash twice with DEPC-PBS-T.
?. [Barcode padlock probe hybridization and ligation]
Reagents for padlock hybridization are combined as in the table below.
?. [Barcode padlock probe hybridization and ligation]
ABCD1Reagent[Stock][Final]1x (µl)2DEPC-H2O223Tth Ligase buffer10x1x54KCl1M0.05 M2.55Formamide100%20%106Padlock Probe0.5 µM10 nM each17BSA20 µg/µl0.2 µg/µl0.58Tth Ligase5 U/µl0.5 U/µl59RNaseH5 U/µl0.4 U/µl410Total50
ABCD1Reagent[Stock][Final]1x (µl)2DEPC-H2O223Tth Ligase buffer10x1x54KCl1M0.05 M2.55Formamide100%20%106Padlock Probe0.5 µM10 nM each17BSA20 µg/µl0.2 µg/µl0.58Tth Ligase5 U/µl0.5 U/µl59RNaseH5 U/µl0.4 U/µl410Total50
Safety precautions: formamide
?. [Barcode padlock probe hybridization and ligation]
DEPC-PBS-T removed and combined reagents added to seal chamber.Chamber is sealed and placed in a humidity chamber.Slide and chamber is incubated for 30 min at 37°C.Enzymes and temperature (see Note 12)Then switched to incubation at 45°C for 1 hour.
37 °C
45 °C
?. [Barcode padlock probe hybridization and ligation]
Then the slide is washed twice with DEPC-PBS-T.
?. [Rolling circle amplification (RCA)]
Reagents for rolling circle amplification (RCA) of padlock probes are combined as in the table below.
?. [Rolling circle amplification (RCA)]
ABCD1Reagents[Stock][Final]1x (µl)2DEPC-H2O333Phi29 buffer10x1x54Glycerol50%5%55dNTPs25 mM0.25 mM0.56BSA20 µg/µl0.2 µg/µl0.57Exonuclease I20 U/µl0.4 U/µl18Phi29 polymerase10 U/µl1 U/µl59Total50
ABCD1Reagents[Stock][Final]1x (µl)2DEPC-H2O333Phi29 buffer10x1x54Glycerol50%5%55dNTPs25 mM0.25 mM0.56BSA20 µg/µl0.2 µg/µl0.57Exonuclease I20 U/µl0.4 U/µl18Phi29 polymerase10 U/µl1 U/µl59Total50
?. [Rolling circle amplification (RCA)]
DEPC-PBS-T removed and combined reagents added to seal chamber.Chamber is sealed and incubated in humidity chamber for4 hours at 37°C OR 30°C overnight.(see Note 13) OR
[37C incubation]
37 °C
30 °C
?. [Rolling circle amplification (RCA)]
After RCA, samples washed twice with DEPC-PBS-T.Samples protected from light.
?. [Anchor primer hybridization]
Reagents for anchor primer hybridization are combined according to table below.
?. [Anchor primer hybridization]
ABCD1Reagents[Stock][Final]1x (µl)2DEPC-H2O24.532x Hybridization buffer2x1x254Anchor Primer10 µM0.1 µM0.55Total50
ABCD1Reagents[Stock][Final]1x (µl)2DEPC-H2O24.532x Hybridization buffer2x1x254Anchor Primer10 µM0.1 µM0.55Total50
?. [Anchor primer hybridization]
Reagents added to chamber and incubated 30 min at RT, in the dark.
[Anchor Incubation]
?. [Anchor primer hybridization]
Slide is washed twice with DEPC-PBS-T.
?. [Sequence by ligation (SBL)]
Reagents for base library hybridization and ligation are combined according to table below.
?. [Sequence by ligation (SBL)]
ABCD1Reagents[Stock][Final]1x (1 µl)2DEPC-H2O39.253T4 Ligase buffer10x1x54ATP25 mM1 mM25Base #A10 µM0.1 µM0.56Base #C10 µM0.1 µM0.57Base #T10 µM0.1 µM0.58Base #G10 µM0.1 µM0.59DAPI100 μg/ml0.5 μg/ml0.2510BSA20 µg/µl0.2 µg/µl0.511T4 DNA Ligase5 U/µl0.1 U/µl112Total50
ABCD1Reagents[Stock][Final]1x (1 µl)2DEPC-H2O39.253T4 Ligase buffer10x1x54ATP25 mM1 mM25Base #A10 µM0.1 µM0.56Base #C10 µM0.1 µM0.57Base #T10 µM0.1 µM0.58Base #G10 µM0.1 µM0.59DAPI100 μg/ml0.5 μg/ml0.2510BSA20 µg/µl0.2 µg/µl0.511T4 DNA Ligase5 U/µl0.1 U/µl112Total50
?. [Sequence by ligation (SBL)]
DEPC-PBS-T removed and combined reagents added to seal chamber.Slide is incubated 1 hour at RT in the dark.
[SBL]
?. [Sequence by ligation (SBL)]
Slide is washed twice with DEPC-PBS-T.
?. [Sequence by ligation (SBL)]
Dip slides into 70% EtOH solution and incubate for 1 min.
[70% EtOH]
?. [Sequence by ligation (SBL)]
Remove the SecureSeal chamber and dip slides in 100% for 1 min(see Note 14)Air dry the slides.
[100% EtOH]
?. [Sequence by ligation (SBL)]
Apply small amount (~10 µL) of mounting medium (Slowfade or similar) and apply a cover slip.(see Note 15)Slide can be stored in the dark and at +4°C for longer term storage before imaging.
?. [Imaging]
Use a standard epifluorescent microscope with 20x objective.Use DAPI staining to focus on section as a reference point to visualize RCA products. For details of microscope setup, check referenced publications.
?. [Imaging]
Acquire images as needed using reference points that can be used for sequential imaging of other bases later on.RCPs are located in different focal planes, therefore, a series of images should be captured at different focal depths. The stacks of images are then merged to a single image using the maximum-intensity projection.
?. [Stripping of anchor and base]
After every round of base imaging, fluorophores and probes need to be stripped of the rolling circle products in order to hybridize the next base.Remove coverslip by dipping slide into 70% EtOH until it slips off.Note: After second and all subsequent rounds of imaging, cover slip can be removed by dipping slide into PBS, dehydration step is not needed if hydrophobic pen is used.
?. [Stripping of anchor and base]
Dip slide into 100% EtOH for 1 min and then air dry the samples.
[100% EtOH]
?. [Stripping of anchor and base]
Use a hydrophobic pen to draw a barrier around section. Alternatively, mount another SecureSeal chamber.
?. [Stripping of anchor and base]
Rehydrate with DEPC-PBS-T.
?. [Stripping of anchor and base]
Combine reagents in the table below for the stripping of anchor and sequence primer.
?. [Stripping of anchor and base]
ABCD1Reagent[Stock][Final]1x (µl)2DEPC-H2O43.53UNG buffer10x1x54BSA20 µg/µl0.2 µg/µl0.55UNG1 U/µl0.02 U/µl16Total50
ABCD1Reagent[Stock][Final]1x (µl)2DEPC-H2O43.53UNG buffer10x1x54BSA20 µg/µl0.2 µg/µl0.55UNG1 U/µl0.02 U/µl16Total50
?. [Stripping of anchor and base]
Reagents added to section and incubated at RT for 30 min.
?. [Stripping of anchor and base]
Sections washed once with DEPC-PBS-T
?. [Stripping of anchor and base]
Sections washed with 100% formamide at RT for 3 min.Repeat an additional two times for a total of 3 times.
[1st Wash]
[2nd Wash]
[3rd Wash]
?. [Stripping of anchor and base]
Sections washed twice with DEPC-PBS-T at RT
?. [Repeat hybridization of anchor and next base.]
Repeat Steps 22-44 for additional required bases.Note: If using hydrophobic pen, chamber will not be used and solutions can just be applied on top of section during incubations.
?. [Image analysis]
For most up to date methodolgy on image analysis, we refer to a recent publication from our group:Probabilistic cell typing enables fine mapping of closely related cell types in situQian X, Harris KD, Hauling T, Nicoloutsopoulos D, Muñoz-Manchado AB, Skene N, Hjerling-Leffler J, Nilsson MNat Methods. 2020 Jan;17(1):101-106.doi: 10.1038/s41592-019-0631-4We also refer to the publication this protocol is based on for image analysis:In situ sequencing for RNA analysis in preserved tissue and cells. Ke R, Mignardi M, Pacureanu A, Svedlund J, Botling J, Wählby C, Nilsson M. Nat Methods. 2013 Sep;10(9):857-60.doi: 10.1038/nmeth.2563(Cell profiler: see Note16)