Targeted ExSeq -- In Situ Sequencing (Illumina Chemistry) 

Preparation of Sample

This protocol assumes that samples have been prepared following the Targeted ExSeq Tissue Preparation protocol, and that a sequencing library has been prepared following the Targeted ExSeq Library Preparation protocol. The padlock probes to generate the library should have been prepared following the Targeted ExSeq Probe Generation protocol.

The protocol assumes that the library has been prepared, and if the amplicons were optionally checked, that the detection oligos have been stripped off as described in the Library Preparation protocol.

Pooling ddNTPs

The dideoxynucleotide set contains four tubes at 10 mM each for each of the dideoxynucleotides.

Combine 10 μL from each tube to form the final 2.5 mM each ddNTP pool.

Preparing Sequencing Reagents from MiSeq Kit

The Illumina in situ sequencing kit reagents are collected from MiSeq reagent kits. Both MiSeq v3 and v2 reagent kits can be used for in situ sequencing. The terminology below is written for the MiSeq v3, with MiSeq v2 terms in parentheses.

Three reagents are collected:

• IMT: Incorporation Mix (v2: IMS, Incorporation Mix)
• USM: Scan Mix (v2: SRE, Scan Mix)
• CMS: Cleavage Mix (v2: CMS, Cleavage Mix)

Defrost a MiSeq v3 reagent kit (either at 4°C or for 1h 0m 0s in a Room temperature water bath). Invert the kit to mix and ensure that the wells are fully melted.

Wipe a clean laboratory wipe over the foil cover of Wells 1, 2, and 4 of the MiSeq Reagent Kit.

Pierce the foil for Wells 1, 2, and 4 with a 1 mL pipette tip.

Use a transfer pipette to transfer reagents in Well 1 (IMT; v2 IMS), Well 2 (USM; v2 SRE), and Well 4 (CMS; v2 CMS) to individual 50 mL tubes.

Aliquot IMT (IMS), USM (SRE), and CMS (CMS) into 250-300 μL aliquots in microcentrifuge tubes, and store at -20°C for up to one month.

In addition, also save the bottle of PR2 provided with the reagent kit.

Gelling Materials from ExSeq Tissue Processing Protocol

The following reagents were prepared in the Targeted ExSeq Tissue Processing Protocol and are used again in this protocol.

1. 10% TEMED
2. 10% APS

Overview of Second Re-embedding

To minimize any movement during the multiple rounds of imaging during in situ sequencing, the sample gel is immobilized to a glass-bottom plate for all subsequent steps. This is accomplished by functionalizing wells of a glass-bottom plate with acryloyl groups (Step 6), and casting a second re-embedding gel to anchor the gel to the plate (Step 7).

Treatment of Glass-Bottom Plate with Bind-Silane

In this step, we treat wells of a 24-well glass-bottom plate with Bind-Silane, a silanization reagent that functionalizes the glass surface with acryloyl groups. The acryolyl groups are incorporated as monomers of the gel formed during the second re-embedding step.

Volumes can be scaled up as is appropriate for 6-well plates.

Cleaning Glass Surfaces

Wash wells briefly with ddH2O, followed by 100% ethanol.

Bind-Silane Reaction

Prepare Bind-Silane reaction mix, scaling up/down total volume as needed.

A	B
Reagent	Volume
Bind-Silane Reagent	10 μL
Ethanol	4000 μL
Glacial Acetic Acid	100 μL
Water	890 μL
Total	5000 μL

Bind-Silane reaction mix

Treat wells of glass-bottom plate for 0h 45m 0s at Room temperature.

Remove bind-silane reaction mix and allow well to dry.

Wash twice briefly with 100% ethanol, then remove ethanol and allow wells to dry completely before use.

Directly proceed to casting second re-embedding gel.

Casting Second Re-embedding Gel

This gelling step is similar to the first re-embedding step from the tissue preparation protocol, but does not have an extended pre-incubation step.

The purpose of the second re-embedding gel is to anchor the sample gel to the glass by incorporating the acryloyl groups on the glass surface into the second re-embedding polyacrylamide gel, which penetrates into the sample gel. The re-embedding gel also supports the sample gel around the sides.

In addition, fluorescent beads are added to the gelling solution to help with the color-correction step in the computational processing of ExSeq data.

Preparation of Re-embedding Gelling Solution and Gelling Chamber

Prepare re-embedding gelling solution as follows. Add reagents in the following order: Water, Acrylamide/Bis, Tris Base, 10% TEMED, 10% APS, TetraSpeck beads. Mix by vortexing after adding each chemical reagent. Do not vortex after adding TetraSpeck beads -- mix by pipetting up and down.

A	B	C	D
Solution	Stock Concentration	Volume	Final Concentration
Acrylamide/Bis 19:1 solution	40% (w/v)	100 μL	~3.8% (w/v) acrylamide; 0.2% (w/v) N-N'-Methylenebisacrylamide
Water		875 μL
Tris Base	1 M	5 μL	5 mM
10% TEMED	10% (w/v)	5 μL	0.05%
10% APS	10% (w/v)	5 μL	0.05%
0.2 μm TetraSpeck beads		10 μL
Total		1000 μL

Re-embedding gelling solution

Pipette a droplet of the re-embedding solution to a well of a Bind-Silane treated plate.

Transfer the sample gel to the well with a paintbrush and place the gel on top of the droplet of re-embedding solution. Orient the sample so that the tissue is on the top of the sample (farthest from the glass)*

Pipette another droplet of the re-embedding solution on top of the sample.

Place a 10 mm circular coverglass on top with tweezers (or larger coverglass if the sample is larger).

Using a pipette, backfill the remaining volume under the coverglass and around the gel.

*The sample orientation can be checked before the second re-embedding by staining with DAPI, imaging the sample, taking note of the in-focus Z-position, then flipping the gel and noting the in-focus Z-position again. The orientation with the higher in-focus Z-position is the orientation to use when gelling.

Nitrogen Purge and Second Re-embedding Gelation

Place 24-well plate into a humidified Tupperware container with two small holes in the lid and close the container.

Purge Tupperware container with nitrogen gas. To do this, start a slow flow of nitrogen gas into a nitrogen line connected to tubing with a blunt needle at the end. The flow rate should be barely perceivable when pointed at skin.

Insert the needle through one of the holes in the lid of the container. Ensure that the other hole is open, allowing for airflow out of the container. Ensure that the container does not immediately bulge or pop-open, which would be indicative of too high of a flow rate.

Purge for 0h 10m 0s.

After purging, withdraw the needle, and seal both holes by covering with tape.

Cast re-embedding by incubating for 1h 30m 0s at 37°C.

Post-Gelation Wash

Remove coverglass with tweezers.

Wash sample with 1X PBS for 0h 20m 0s at Room temperature.

--- Pause Point ---

The plate can be stored at 4°C for up to two weeks.

Overview of Sequencing Preparation

In this step, we prepare the glass-anchored sample for in situ sequencing with the Illumina chemistry. There are two primary steps here: (1) capping terminal 3' DNA ends of cellular DNA with dideoxy nucleotides; and (2) hybridization of the Illumina sequencing primer.

Dideoxy Capping of 3' DNA Strands

To minimize background from base addition to exposed 3' ends of cellular DNA during the Illumina incorporation, Terminal deoxynucleotidyl transferase (TdT) is used to add dideoxynucleotides to block further extension.

Pre-incorporation

Prepare TdT pre-incorporation mix and add to sample for 0h 20m 0s atRoom temperature.

A	B	C	D
Reagent	Stock Concentration	Volume	Final Concentration
TdT buffer	10X	20 μL	1X
CoCl2	2.5 mM	20 μL	250 μM
ddNTP	2.5 mM each ddNTP	4 μL	50 μM each ddNTP
ddH2O		156 μL
Total		200 μL

TdT pre-incorporation mix

TdT Capping

Prepare TdT reaction mix, add to sample, and incubate for 1h 30m 0s at 37°C.

A	B	C	D
Reagent	Stock Concentration	Volume	Final Concentration
TdT buffer	10X	20 μL	1X
CoCl2	2.5 mM	20 μL	250 μM
ddNTP	2.5 mM each ddNTP	4 μL	50 μM each ddNTP
TdT enzyme	20 U/μL	4 μL	0.4 U/μL
ddH2O		152 μL
Total		200 μL

TdT Reaction Mix

PBS Washes

Wash gels with 1X PBS for 0h 10m 0s at Room temperature.

Sequencing Primer Hybridization

In this step, the sequencing primer is hybridized to the amplicons. The sequence for the Illumina sequencing primer used for the padlock probes designed in the probe design protocol is TCT CGG GAA CGC TGA AGA CGG C, and should be ordered with HPLC purification (see materials).

Sequencing Primer Hybridization

Prepare the sequencing primer hybridization mix by diluting the 100 μM sequencing primer stock by 1:40 in 4X SSC, forming a 2.5 μM sequencing primer mix.

Incubate gel with the sequencing primer hybridization mix in 4X SSC for 1h 0m 0s at 37°C.

SSC Washes

Wash gels with 4X SSC for 0h 10m 0s at 37°C.

PR2 Washes

Wash gel with PR2 buffer for 0h 10m 0s at Room temperature.

Overview

The next three sections cover the process of in situ Illumina sequencing-by-synthesis.

The first step is elongation, the addition of a new base (Step 12). The second step is imaging (Step 13). The third step is cleavage (Step 14). These steps are repeated for each base of the barcode. For the barcode structure described by this protocol, a total of seven rounds of sequencing are required.

Overview

In this step, the next base of the barcode is synthesized. This base is complementary to the template base of the barcode in the amplicon.

Incorporation Mix Pre-incubation

Prepare ISS Incorporation Mix: 0.5X IMT and 2.5 mM MgCl2 in 1X Taq Reaction Buffer.

A	B	C	D
Reagent	Stock Concentration	Volume	Final Concentration
IMT (from Illumina kit)	1X	500 μL	0.5X
MgCl2	25 mM	100 μL	2.5 mM
Taq Reaction Buffer	10X	100 μL	1X
ddH2O		300 μL
Total		1000 μL

ISS Incorporation Mix Wash sample with ISS Incorporation Mix for 0h 15m 0s at Room temperature.

Incorporation

Treat sample with ISS Incorporation Mix for 0h 10m 0s at50°C.

Post-incorporation Washes

Wash with 2% Zwittergen (w/v) in PR2 for 0h 15m 0s at50°C.

If harsher washing is needed, add 0.1 M MES buffer, pH 6.

Then, wash with PR2 for 0h 15m 0sat 50°C.

DAPI Staining

Stain with DAPI (1 mg/L) in PR2 for 0h 15m 0s atRoom temperature.

Overview

In this step, we transfer the sample to the Illumina imaging buffer and image the current round of the barcode.

Washing with Imaging Buffer

Wash samples with USM buffer (from Illumina kit) for 0h 15m 0s at Room temperature.

The sample is now ready for imaging. Keep the sample in USM buffer.

Overview of Imaging

The specifics of imaging vary significantly from microscope to microscope.

In general, users will be performing tiled array imaging, where each tile is a multicolor Z-section.

We recommend a 2% overlap between tiles to minimize photobleaching of amplicons at the edges of tiles. We recommend a spacing of 0.4 μm between adjacent Z-sections, and we recommend setting up the image acquisition to image all Z-places for a color channel before moving on to the next color channels. Imaging should be performed from longest to shortest excitation wavelength. Take care when setting up the image acquisition to ensure that the sample will be fully covered in Z across the entire XY region, since a small sample tilt can result in a significant fraction of the sample being out of the imaged volume.

This protocol is optimized for imaging on a Dragonfly spinning disk confocal microscope, using a Nikon 40X water immersion, long working distance, NA 1.15 objective (MRD77410). The key elements are shown in the following table. All lasers are set to 100% output power.

A	B	C	D	E
Channel Description	Excitation (nm)	Excitation Laser Power (mW)	Emission Range (nm)	Exposure time (ms)
Template Base G	685	30	705-845	400
Template Base T	640	160	663-737	200
Template Base A	561	150	575-590	200
Template Base C	488	150	500-550	400
DAPI	405	100	440-460	300

Summary of imaging parameters.

If a 685 laser is not available, the 640 laser can be used to excite template base G (added base C), and the signal can be separated from Template Base T (added base A) using a 775/140 emission filter (or similar). This will have slightly lower signal to background.

Overview

In this step, the Illumina reversible terminator is unblocked, allowing for synthesis of the next base. The fluorophore added with the last base is also cleaved off the sequencing product.

Post-Imaging Washes

Wash with PR2 buffer for 0h 10m 0s at Room temperature.

Pre-incubation with Cleavage Buffer

Wash with CMS (cleavage buffer from Illumina Kit) for 0h 10m 0s at 37Room temperature.

Cleavage

Treat sample with CMS (cleavage buffer from Illumina Kit) for 0h 20m 0s at 50°C.

Post-Cleavage Washes

Wash with PR2 buffer for 0h 10m 0s at 50.

Then, wash with PR2 buffer for 0h 10m 0s at 37Room temperature.

Iteration

If more bases need to be sequenced, .

Recovery of Antibody Staining

If antibody staining with a biotinylated secondary antibody was previously performed in the tissue processing step, this section recovers and images that signal.

For imaging the antibody staining: if sub-voxel resolution registration to the in situ sequencing data will be performed, skip the final cleavage step after the last base of sequencing and image the antibody staining signal with the last base of sequencing. The puncta signal can be used in the computational processing to co-register this dataset.

If another feature (i.e. DAPI) will be used to coregister the antibody staining, the cleavage can be performed before antibody staining signal recovery.

[Only if Final Cleavage is Skipped] PR2 Wash

Wash the samples with PR2 buffer for 0h 30m 0s at Room temperature.

Stain with Fluorophore-Labeled Streptavidin

Incubate the samples with 10 μg/mL fluorophore-labeled Streptavidin (i.e. Streptavidin-Alexa 488) in PR2 for at 4°C.

The next day, wash samples with PR2 for 0h 30m 0s at Room temperature.

DAPI Staining

Stain with DAPI (1 mg/L) in PR2 for 0h 15m 0s atRoom temperature.

Washing with Imaging Buffer

Wash samples with USM buffer (from Illumina kit) for 0h 15m 0s at Room temperature.

Add USM buffer to the sample again.

Imaging

The precise imaging parameters depend on the specifics, but the imaging protocol for sequencing may serve as a template.