TITLE: Targeted ExSeq -- In Situ Sequencing (Illumina Chemistry) 
AUTHORS: Yi  Cui, Anubhav    Sinha, Ed  Boyden

[DESCRIPTION]

  
This protocol accompanies Expansion Sequencing (ExSeq), and describes the process of performing in situ sequencing with the Illumina chemistry.

The steps here are an extension of the protocol used in the original paper and are optimized for reading longer barcodes of up to seven bases. For shorter barcodes (less than four bases) in brain tissue, we recommend using the experimental protocol from the paper (with the imaging conditions as described in this protocol) as it is simpler and slightly faster.

There are two stages to this protocol: (1) sample preparation (steps 5-10), involving anchoring the sample to a glass-bottom plate and preparing the sample for sequencing; and (2) in situ sequencing using the Illumina sequencing-by-synthesis chemistry (steps 11-15). After in situ sequencing is complete, there is an optional antibody staining recovery step (step 16) if antibody staining was performed during tissue preparation.

The relationship between template bases and fluorophores is shown in Fig. 1A. Note that the added base (that bears the fluorophore) is the complementary base, i.e. the modified dCTP from the Illumina reagents bears the most redshifted dye. The specific length 7, Hamming distance 3 logical barcode library used in these protocols is shown in Fig. 1B, with the specific readout strategy for Illumina in situ sequencing shown in Fig. 1C.

The process of Illumina sequencing-by-synthesis in situ is shown in Fig. 1D. There are three key steps: an incorporation step, in which the next base of the barcode is synthesized; an imaging step, in which the sample is imaged on a spinning disk confocal microscope; and a cleavage step, in which the dye is cleaved, and the reversible terminator is unblocked, enabling the next round of synthesis to be performed.

This protocol was used to profile human metastatic breast cancer biopsies as a part of the Human Tumor Atlas Pilot Project.

[BEFORE_START]
Before starting, prepare stock solutions/reagents as described in steps 1-4 of the protocol.

[GUIDELINES]
This protocol follows the Tissue Preparation and Library Preparation protocols; those guidelines also apply here.

[STEPS]
SECTION: Illumina Sequencing: Cleavage
15. Iteration

If more bases need to be sequenced,  .
SECTION: Preparation
1. 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.

SECTION: Second Re-embedding Gel
5. 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).
SECTION: TdT treatment &amp; Primer Hybridization
8. 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.
SECTION: TdT treatment &amp; Primer Hybridization
9. 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.

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10. 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).
SECTION: TdT treatment &amp; Primer Hybridization
10.1. 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 60 min at 37 °C.
SECTION: TdT treatment &amp; Primer Hybridization
10.2. SSC Washes

Wash gels with 4X SSC for 10 min at 37 °C.
SECTION: TdT treatment &amp; Primer Hybridization
10.3. PR2 Washes

Wash gel with PR2 buffer for 10 min at Room temperature.
SECTION: In Situ Illumina Sequencing: Overview
11. 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.
SECTION: TdT treatment &amp; Primer Hybridization
9.1. Pre-incorporation

Prepare TdT pre-incorporation mix and add to sample for 20 min atRoom temperature.
 
TdT buffer, 10x20CoCl2, 2.5 mM20ddNTP, 2.5 mM4H2O156

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9.3. PBS Washes

Wash gels with 1X PBS for 10 min at Room temperature.

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9.2. TdT Capping

Prepare TdT reaction mix, add to sample, and incubate for 90 min at 37 °C.
 
     ABCD1ReagentStock ConcentrationVolumeFinal Concentration2TdT buffer10X20 μL1X3CoCl22.5 mM20 μL250 μM4ddNTP2.5 mM each ddNTP4 μL50 μM each ddNTP5ddH2O156 μL6Total200 μL

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6. 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.
SECTION: Second Re-embedding Gel
6.1. Cleaning Glass Surfaces

Wash wells briefly with ddH2O, followed by 100% ethanol.
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6.2. Bind-Silane Reaction

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

 

Treat wells of glass-bottom plate for 45 min 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.
SECTION: Second Re-embedding Gel
7. 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.
SECTION: Second Re-embedding Gel
7.1. 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.
 
 

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.
SECTION: Second Re-embedding Gel
7.2. 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 10 min.

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

Cast re-embedding by incubating for 90 min at 37 °C.
SECTION: Second Re-embedding Gel
7.3. Post-Gelation Wash

Remove coverglass with tweezers.

Wash sample with 1X PBS for 20 min at Room temperature.

--- Pause Point ---

The plate can be stored at 4 °C  for up to two weeks.
SECTION: Illumina Sequencing: Elongation
12. 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.
SECTION: Illumina Sequencing: Elongation
12.1. Incorporation Mix Pre-incubation

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

 
Wash sample with ISS Incorporation Mix for 15 min at Room temperature.

SECTION: Illumina Sequencing: Elongation
12.2. Incorporation

Treat sample with ISS Incorporation Mix for 10 min at50 °C.
SECTION: Illumina Sequencing: Elongation
12.3. Post-incorporation Washes

Wash with 2% Zwittergen (w/v) in PR2 for 15 min at50 °C.

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

Then, wash with PR2 for 15 minat 50 °C.
SECTION: Illumina Sequencing: Elongation
12.4. DAPI Staining

Stain with DAPI (1 mg/L) in PR2 for 15 min atRoom temperature.
SECTION: Illumina Sequencing: Cleavage
14. 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.
SECTION: Illumina Sequencing: Cleavage
14.1. Post-Imaging Washes

Wash with PR2 buffer for 10 min at  Room temperature.

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14.2. Pre-incubation with Cleavage Buffer

Wash with CMS (cleavage buffer from Illumina Kit) for 10 min at 37 Room temperature.
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14.3. Cleavage

Treat sample with CMS (cleavage buffer from Illumina Kit) for 20 min at 50 °C.
SECTION: Illumina Sequencing: Cleavage
14.4. Post-Cleavage Washes

Wash with PR2 buffer for 10 min at 50 10.

Then, wash with PR2 buffer for 10 min at 37 Room temperature.
SECTION: [OPTIONAL] Antibody Signal Recovery and Imaging
16. 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.
SECTION: [OPTIONAL] Antibody Signal Recovery and Imaging
16.1. [Only if Final Cleavage is Skipped] PR2 Wash

Wash the samples with PR2 buffer for 30 min  at Room temperature.
SECTION: [OPTIONAL] Antibody Signal Recovery and Imaging
16.2. 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 30 min at Room temperature.
SECTION: [OPTIONAL] Antibody Signal Recovery and Imaging
16.3. DAPI Staining

Stain with DAPI (1 mg/L) in PR2 for 15 min atRoom temperature.
SECTION: [OPTIONAL] Antibody Signal Recovery and Imaging
16.4. Washing with Imaging Buffer

Wash samples with USM buffer (from Illumina kit) for 15 min at Room temperature.

Add USM buffer to the sample again.
SECTION: [OPTIONAL] Antibody Signal Recovery and Imaging
16.5. Imaging

The precise imaging parameters depend on the specifics, but the imaging protocol for sequencing may serve as a template. 
SECTION: Illumina Sequencing: Imaging
13. Overview

In this step, we transfer the sample to the Illumina imaging buffer and image the current round of the barcode.
SECTION: Illumina Sequencing: Imaging
13.1. Washing with Imaging Buffer

Wash samples with USM buffer (from Illumina kit) for 15 min at Room temperature.

The sample is now ready for imaging. Keep the sample in USM buffer.
SECTION: Illumina Sequencing: Imaging
13.2. 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. 
G488500-550T561575-590A640663-737C685705-845
 
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.
SECTION: Preparation
2. 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.
SECTION: Preparation
3. 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 60 min 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.
SECTION: Preparation
4. 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.

10% TEMED
10% APS