TITLE: Light-Seq
AUTHORS: Jocelyn Y. Kishi, Ninning Liu, Emma R. West, Kuanwei Sheng, Jack J. Jordanides, Matthew Serrata, Constance L. Cepko, Sinem K. Saka, and Peng Yin

[DESCRIPTION]
We present Light-Seq, an approach for multiplexed spatial indexing of intact biological samples using light-directed DNA barcoding infixed cells and tissues followed by ex situ sequencing. Light-Seq combines spatially targeted, rapid photocrosslinking of DNA barcodes onto complementary DNAs in situ with a one-step DNA stitching reaction to create pooled, spatially indexed sequencing libraries. This light-directed barcoding enables in situ selection of multiple cell populations in intact fixed tissue samples for full-transcriptome sequencing based on location, morphology or protein stains, without cellular dissociation. Applying Light-Seq to mouse retinal sections, we recovered thousands of differentially enriched transcripts from three cellular layers and discovered biomarkers fora very rare neuronal subtype, dopaminergic amacrine cells, from only 4–8 individual cells per section. Light-Seq provides an accessible workflow to combine in situ imaging and protein staining with next generation sequencing of the same cells, leaving the sample intact for further analysis post-sequencing.

[STEPS]
SECTION: Before Starting
2. This protocol is for selective sequencing of cells within fixed tissue sections that have been sectioned on to a coverslip or microscope slide. 

Clean workspace (bench, pipettes, etc.) with ethanol before starting. When the protocol calls for water, always use UltraPure water. All reagents should be molecular-grade and RNAse free. All reactions with enzymes are prepared on ice.

Key Equipment:
Microscope
Light-Seq requires an optical system that can focus UV light onto specific regions of interest. In the publication, we employ a wide field microscope with a 365 nm LED and a digital micromirror device (DMD) to accomplish this, but other systems are also amenable. We have also tested a point-scanning confocal microscope with a 405 nm laser. We recommend consulting your microscopy core or representative to discuss the best solution at your institution.

Flat-top Thermocycler
The first few steps of the protocol require thermal cycling and incubations of tissue slides. For this, we recommend the Eppendorf® Mastercycler® nexus Flat Thermal Cyclers (VWR Cat No. 71003-568).



SECTION: A-tailing
6. After in situ RT, A-tailing is performed to add a polyA tail to the 3’ end of newly transcribed cDNAs, providing a handle on the 3’ end for downstream amplification. The A-tailing reaction is isothermal at 37C, and can be performed in a flat-top thermocycler or in a hybridization oven. Once cDNAs are A-tailed, immunofluorescent staining can optionally be performed to identify cells before barcoding. 

A-tailing Reagents 
   ReagentsSupplierCat. No.Deoxynucleotide (dNTP) Solution Set- 25 μmol each at 100 mM (just needdATP)NEBN0446S2′,3′-Dideoxyadenosine 5′-Triphosphate, 100 mM solution(ddATP)Sigma AldrichGE27-2051-01Terminal Transferase - 2,500 unitsNEBM0315LThermoPol® Reaction Buffer PackNEBB9004STWEEN® 20Sigma AldrichP9416- 50MLPBSInvitrogenAM9625
 
Perform A-tailing, in situ
1. Create aliquots of 10 mM dATP (20 µL aliquots) and 25 mM ddATP (~3 µL aliquots). Store at -20C.
2. Dilute ddATP stock to 250 μM (1:100 dilution from 25 mM stock).
3. Create A-tailing Master Mix on ice:
 

4. Wash wells once with 0.1% PBS-Tw.
5. Aspirate, add 50 μL A-tailing Master Mix and incubate for 45min at 37C. If using Grace Bio-Labs chambers, be sure to cover the holes with a coverslip or stickers to prevent evaporation.
6. Wash 3 x 1min in 0.1% PBS-Tw. 

Optional Pause Point: Store at 4C overnight before barcoding.
SECTION: Light-Directed Barcoding of cDNAs
8. Light-Directed Barcoding of Select Cells for Sequencing
Light-directed barcoding is performed sequentially for different regions of interest/cell populations within the same sample. For sequencing three regions, three rounds of barcoding are performed with a unique barcode sequence used in each round. The three barcodes used in the publication are listed in the table below, and in Supplementary Table 6. 

  
 Light-Seq Barcode Sequences
     Sequence name      Purpose      Sequence        GATE.D12.B1      Barcode sequence 1 - Cy5   labeled barcode strand.      GGAGTTGGAGTGAGTGGATGAGTGATGDDDDDDDDDDDDTATGGATGAGTTATATAACTCA[cnvK]TCGTGTAAAT[Cy5-3]        GATE.D12.B2      Barcode sequence 2 - Cy3   labeled barcode strand.      GGAGTTGGAGTGAGTGGATGAGTGATGDDDDDDDDDDDDGTTAGGTGAGTTATATAACTCA[cnvK]TCGTGTAAAT[Cy3-3]        GATE.D12.B3      Barcode sequence 3 -Fluorescein   (FITC) labeled barcode strand.      GGAGTTGGAGTGAGTGGATGAGTGATGDDDDDDDDDDDDAGGGTATGAGTTATATAACTCA[cnvK]TCGTGTAAAT[Fl-3]   

Barcoding Reagents
   ReagentsSupplierCat. No.Dextran Sulfate 50% solutionSigma AldrichS4030Salmon-sperm DNAThermo FisherAM96805 M NaClInvitrogenAM9760GTWEEN® 20Sigma AldrichP9416- 50MLPBSInvitrogenAM9625FormamideInvitrogenAM9342UltraPure WaterInvitrogen10977023Barcode DNA Oligos (see sequences table)GeneLink

Light-Directed Barcoding of cDNAs
1. Prepare barcoding solutions:
        Stringent 60% Wash (0.1% PBS-Tw + 60% formamide):
        4 mL 10X PBS
        24 mL 100% formamide
        12 mL UltraPure H2O
        40 μL Tween-20
        Note: Store at 4C for use later during barcoding.

        High-Salt Wash (1X PBS + 1 M NaCl + 0.1% Tween- 20):
        4 mL 10X PBS
        8 mL 5 M NaCl
        28 mL UltraPure H2O
        40 μL Tween-20
        Note: Store at room temperature for use later during barcoding.

        Hybridization Master Mix
 

2. Add barcoding strands to Hybridization Master Mix to create Barcoding Solutions:

Barcode 0 Solution (per 50 uL well - make excess!):
1.25 μL of 10 µM Barcode Strand (GATE.D12.B0)
48.75 μL hybridization master mix

Barcode 1 Solution (per 50 uL well - make excess!):
1.25 μL of 10uM Barcode Strand (GATE.D12.B1)
48.75 μL hybridization master mix

Barcode 2 Solution (per 50 uL well - make excess!):
1.25 μL of 10uM Barcode Strand (GATE.D12.B2)
48.75 μL hybridization master mix

3. Replace liquid in each well with High-Salt Wash.
4. Aspirate to remove all liquid, add Barcode 0 Solution. Incubate for 30 min at room temperature to allow barcodes to hybridize to cDNA 5' overhangs. Cover slide with foil to protect from light.
5. Aspirate to remove all liquid, then wash 3 x 1 min in High-Salt Wash. 
6. Replenish the well with High Salt Wash. Ensure that the sample is completely covered in liquid. 
7. Bring sample to the microscope to perform imaging and light-directed barcoding. Use bright-field or histological stain to visualize cells/region of interest. Be sure to avoid using the UV channel for imaging stains, as this will induce off-target photo-crosslinking of barcodes outside of the region of interest. 
        Note: If evaporation occurs during barcoding, add more High Salt Wash.
8. Perform light-directed barcoding.
  
9. Remove slide from microscope and wash 8 times consecutively in Stringent 60% Wash. 
Note: For applications where very few cells are targeted (<50 cells), increasing the number of stringent washes will likely help reduce background. We recommend up to doubling the number of stringent washes after each round of barcoding.
10. Wash 2 x 2 min in High-Salt Wash, then add fresh High-Salt Wash.
11. For additional barcoding rounds to sequence additional cell populations, repeat steps 3-10 with Barcode 1 Solution and Barcode 2 Solution.

  
12. Transfer to 0.1% PBS-Tween and optionally image barcoded cDNAs to visualize.

Optional Pause Point : Store at 4C overnight in a humidified chamber. If using Grace Bio-Labs chambers, be sure to cover the holes with a coverslip or stickers to prevent evaporation.

 
  
SECTION: Displacement of cDNAs
9. Now that cDNAs are barcoded and A-tailed, they must be extracted for library preparation and sequencing. To do this, the RNA template is digested by mild RNaseH treatment, which degrades the RNA bound to the barcoded cDNAs in situ, liberating the barcoded cDNAs for collection.

Note: Use low retention tips.

  
The displaced barcoded-cDNAs contain a photocrosslink, preventing normal amplification by PCR. Therefore, the Cross-Junction Synthesis reaction creates a single stitched product strand containing the DNA barcode and the cDNA sequence, producing a single amplifiable strand for library preparation and sequencing.

  

Displacement Reagents 
   ReagentsSupplierCat. No.ThermoPol® Reaction Buffer PackNEBB9004SRNase H - 1,250 unitsNEBM0297LUltraPure WaterInvitrogen10977023

Cross-Junction Synthesis Reagents 
   ReagentsSupplierCat. No.Bst DNA Polymerase, Large Fragment- 8,000 units (includes 10XTP and100mM MgSO4)NEBM0275LThermoPol® Reaction Buffer PackNEBB9004SUltraPure WaterInvitrogen10977023Deoxynucleotide (dNTP) Solution Mix - 8 μmol at 10mM eachNEBN0447S
 
Cross-Junction Synthesis Primer
     NameDescriptionSequenceSupplierPurificationGATC.20TPrimer for Cross-Junction SynthesisGAGAATGTGAGTGAAGATGTATGGTGATTTTTTTTTTTTTTTTTTTTIDTHPLC
 

Displace and collect cDNAs separately for each well
1. Dilute cross-junction synthesis primer GATC.20T.p in UltraPure water to make 1 uM stock.
            5 µL 10 µM primer (GATC.20T.p) 
            45 µL UltraPure water 
2. Make Displacement Mix.
 
3. Aspirate liquid from wells and add 50 μL of Displacement Mix to each well. 
4. Cover the well holes with a drop of Mineral Oil and incubate at 37C for 45min.
            NOTE: If using Grace Bio-Labs chambers, we recommend Mineral Oil to minimize loss of solution during         displacement. 
5. While samples are incubating, prepare a separate collection tube for each well by adding 1.2 μL of 1 μM primer (GATC.20T.p) to a low bind PCR tube. 
6. After the incubation, pipette well solution up and down 5-7 times in the well without creating bubbles.
7. Collect all liquid from the well in the corresponding collection tube, prepared in Step 5. Repeat for all wells using fresh tips for each.
8. Wash wells 3 times with 0.1% PBS-Tw. Move to 4C in a humidified chamber for storage, ensuring the wells are covered to prevent evaporation.
9. Heat inactivate the collection tubes in a thermocycler at 75C for 20min.
10. Move tubes to ice and proceed to Cross-Junction Synthesis.

Optional: Samples can be imaged after displacement to verify that the fluorescent barcode signal is no longer present. At this point, follow-up stains can be performed on the sample including antibody stains, H&E, etc. 

  


Cross-Junction Synthesis
1. Add 8.16 μL Cross-Junction Synthesis Mix to each tube:
 
2. Vortex and spin briefly.
3. Incubate in a thermocycler for:
                 37C for 30 mins
                 80C for 20 mins
4. The resulting products are CJS Samples. Proceed to qPCR to amplify CJS Samples.

Optional Pause Point : Store at -20C overnight.
SECTION: qPCR Amplification
11. After the test qPCR on a small amount of sample, a PCR is performed on all of the  CJS Samples to amplify the entire sample for library preparation and sequencing. The cycle number for amplification is chosen based on the test qPCR in the previous step, to prevent over-amplification. Each Cross-Junction Synthesis product (1 per sample well) is amplified in a separate tube. This protocol is identical to the prior qPCR, but scaled to amplify the entire sample and with a reduced number of cycles.

All of the sample should be amplified. However, most qPCR machines have a limit of 50 uL/reaction. Therefore, the full volume should be split into multiple PCRs to ensure accurate amplification.

Amplification of Full Samples
1. Briefly vortex and spin CJS Samples.
2. Measure the volume of CJS sample in each tube and use to calculate volume of qPCR master mix needed for each sample (1:1 ratio of qPCR master mix : CJS sample).
        Note: Ensure that PCR volumes do not exceed the limits for your machine and otherwise, split into multiple tubes.
3. Dilute Sybr Green I dye in water to make 5x solution (e.g. dilute 10,000X stock twice, 1:100 in water, then 1:20 in water).
4. Create qPCR master mix:
 
5. Add the entire remaining sample with equal parts master mix to each tube.
6. Quickly vortex and spin down reactions.
7. Place into qPCR machine for exactly XX cycles and run the Full qPCR Machine Thermocycler Program. 
        
Full qPCR Machine Thermocycler Program
        98C for 3 minutes
        XX cycles of:
            98C for 20 seconds
            60C for 30 seconds
            72C for 2 minutes
            Plate read
        72C for 5 minutes
        Hold at 4C
*XX is based on the amplification curves from the test qPCR. This number will likely range between 16 and 25.
8. The resulting products are Amplified Samples.
SECTION: PCR
None. 1.  Vortex samples from cross-junction synthesis reaction

2.  Create 1:10 dilutions in H2O: 18uL H2O + 2uL sample

3.  Dilute Sybr Green I dye 2,000x in water to make 5x solution (e.g. 1 in 100 dilution, then 1 in 20)

4.  Create PCR master mix:
 

5. Gently mix (flick and invert tube a few times, or mix up and down with pipette) and spin down master mixes

6. Add 49 uL of master mix to appropriate wells

7. Add 21 uL of appropriate reaction/water to wells

8. Gently mix reactions up and down with pipette tips

9. Split each into two 35 uL reactions

10. Place into PCR machine for exactly 26 cycles and run the following program:

Step 1: 
    95°C for 3 minutes
Step 2 (26 cycles):
    95°C for 30 seconds
    54°C for 60 seconds
    68°C for 2 minutes and 30 seconds
    Plate read
Step 3: 
    68°C for 5 minutes
    Hold at 4°C
   
SECTION: Library Prep: Tagmentation and Amplification
None. 

Library Preparation for Next-Generation Sequencing: Tagmentation and Amplification for Illumina Sequencing  
SECTION: Library Prep: Tagmentation and Amplification
None. 
Purify samples with 1.2X Ampure beads, elute in 30uL ultrapure water

Purify with Ampure XP beads at 1.2X bead to sample ratio:

1. Mix 70uL sample with 84uL beads

2. Mix well by pipetting (20x), incubate at RT for 5 min. DO NOT
overextend this step

3. Place on Mag stand for 2 min (should start clearing in <1 min)

4. Add 200 ul of wash buffer (~80% ethanol)

5. Let sit for 30 sec x3

7. Discard ethanol and aspirate remainder with a small 10 ul tip

8. Air dry for 2 min (Do NOT air dry for more than 5 min, especially if
fragments are over 10kb, which can lead to them sticking to beads if
over-dried)
    You can see if the beads change color during this process
    During this time you can try to spin down VERY briefly to get rid of any residual ethanol

9. Add 30 ul of Ultrapure water

10. Mix well by pipetting (20x) and let set for 2 min

11. Place on Mag stand for 1 min. Collect supernatant
SECTION: Library Prep: Tagmentation and Amplification
None. Measure concentrations with Qubit HS dsDNA assay

Note: use clean plastic tubes for all solutions. Do not use glass

Preparing Qubit working solution:
Dilute the Qubit HS Reagent (Component A) 1:200 in the HS buffer (Component B). Will need 400uL working solution for two standards + 200uL per sample (e.g., for 1 samples and 2 standards, mix 594uL HS Buffer (Comp B) + 6uL HS Reagent (Comp A)). Mix well by vortexing.

Create standards 1 and 2:

Standard 1:
190 ul Working solution
10 ul of standard
(Standard 1 should have 0 ng/ul DNA)

Standard 2:
190 ul Working solution
10 ul of standard
(Standard 2 should have 10 ng/ul DNA)

Create samples solutions:

For samples:
198 ul of working solution 
2 ul of sample

Mix standards and samples well by vortexing.

Measure concentrations with Qubit Fluorometer.



SECTION: Library Prep: Tagmentation and Amplification
None. Perform tagmentation

1. (Pre-set PCR machine for the 5 minute 55°C incubation followed by 10°C)

2. To each PCR tube (on ice):

    a. Add 10uL of tagment DNA buffer (TD)

    b. Add 2ng of dsDNA and water up to 5uL (see separate excel sheet for calculations)

    c. Add 5uL of tagmentation enzyme (Amplicon tagment mix - ATM) to lid of tube

3. Quickly spin down tubes, vortex, and spin down

4. Place into PCR machine and incubate at 55°C for EXACTLY 5 minutes

5. Immediately stop reactions with 5uL stop buffer (Neutralize Tagment buffer - NT), hold on ice


SECTION: Library Prep: Tagmentation and Amplification
None. Perform amplification with Nextera and custom primers 

(Recommended: pre-set PCR machine with PCR protocol below)

1. To each tube:

    a. Add 6.5uL water

    b. Add 1.75uL of standard Nextera (i7) primer

    c. Add 1.75uL of custom Nextera (GATE, i5) primer

    d. Add 15uL PCR mix (NPM PCR master mix)

2. Vortex and spin down reactions

3. Start PCR program:

Step 1:
    72°C for 3 minutes
    95°C for 15 seconds
Step 2 (12 cycles):
    95°C for 15 seconds
    55°C for 15 seconds
    72°C for 40 seconds
Step 3:
    72°C for 1 minute
    Hold at 10°C

4. Purify samples with 0.9X Ampure beads, elute in 30uL ultrapure water 

5. Measure concentrations with Qubit assay
SECTION: Library Preparation for Illumina Sequencing
12. Light-Seq uses conventional tagmentation-based library preparation for Illumina sequencing, but with custom primers for the secondary PCR and for Read 1/i5 sequencing. The custom primers are necessary to specifically enrich for and sequence amplicons containing the light-directed barcode sequences. The first step is bead purification of the amplified libraries for each well, followed by tagmentation, secondary PCR amplification, and a second bead purification.

Library Preparation Reagents
   ReagentsSupplierCat. No.Magnetic Separator10X Genomics120250Ampure XP BeadsBeckmanA63881Ethyl alcohol, pure (200 proof)Sigma AldrichE7023-1LQubit™ 1X dsDNA High Sensitivity (HS) and Broad Range (BR) Assay KitsInvitrogenQ33231Nextera XT Library Preparation KitIlluminaFC-131-1096UltraPure WaterInvitrogen10977023
 
Library Preparation DNA Oligos
These primer sequences are used for unique indexing of samples for pooled sequencing. For each sample, a unique pair of S50X and Next.N70X are required. You do not need to order all primers, only enough pairs to uniquely index your samples of interest. Because the custom i5 index primer does not work well on all Illumina machines, we highly recommend each sample be prepared with a unique i7 index. We hope to adjust our recommendations on this front soon, so stay tuned for updated protocols.
     NameDescriptionSequenceSupplierPurificationS502.GATEPrimer for library prep - i5 (barcode) side.AATGATACGGCGACCACCGAGATCTACACCTCTCTATCGCCGGAGTTGGAGTGAGTGGATGAGTGATGIDTHPLCS503.GATEPrimer for library prep - i5 (barcode) side.AATGATACGGCGACCACCGAGATCTACACTATCCTCTCGCCGGAGTTGGAGTGAGTGGATGAGTGATGIDTHPLCS505.GATEPrimer for library prep - i5 (barcode) side.AATGATACGGCGACCACCGAGATCTACACGTAAGGAGCGCCGGAGTTGGAGTGAGTGGATGAGTGATGIDTHPLCS506.GATEPrimer for library prep - i5 (barcode) side.AATGATACGGCGACCACCGAGATCTACACACTGCATACGCCGGAGTTGGAGTGAGTGGATGAGTGATGIDTHPLCS507.GATEPrimer for library prep - i5 (barcode) side.AATGATACGGCGACCACCGAGATCTACACAAGGAGTACGCCGGAGTTGGAGTGAGTGGATGAGTGATGIDTHPLCS508.GATEPrimer for library prep - i5 (barcode) side.AATGATACGGCGACCACCGAGATCTACACCTAAGCCTCGCCGGAGTTGGAGTGAGTGGATGAGTGATGIDTHPLCNext.N701(Normal) primer for library prep - i7 side.CAAGCAGAAGACGGCATACGAGATTCGCCTTAGTCTCGTGGGCTCGGIDTHPLCNext.N702(Normal) primer for library prep - i7 side.CAAGCAGAAGACGGCATACGAGATCTAGTACGGTCTCGTGGGCTCGGIDTHPLCNext.N703(Normal) primer for library prep - i7 side.CAAGCAGAAGACGGCATACGAGATTTCTGCCTGTCTCGTGGGCTCGGIDTHPLCNext.N704(Normal) primer for library prep - i7 side.CAAGCAGAAGACGGCATACGAGATGCTCAGGAGTCTCGTGGGCTCGGIDTHPLCNext.N705(Normal) primer for library prep - i7 side.CAAGCAGAAGACGGCATACGAGATAGGAGTCCGTCTCGTGGGCTCGGIDTHPLCNext.N706(Normal) primer for library prep - i7 side.CAAGCAGAAGACGGCATACGAGATCATGCCTAGTCTCGTGGGCTCGGIDTHPLC

Bead purification of Amplified Samples
NOTE: Each well/replicate is processed separately .
Make 50 mL of 80% Ethanol, diluted in UltraPure water.
For each well, combine 80 µL Amplified Sample with 96 µL Ampure XP Beads in a new PCR tube.
Mix well by pipetting (incubate at room temperature for 5 min). DO NOT overextend this step.
Place on Magnetic Separator Stand for 2 min.
Discard supernatant. Wash 3 x 30 sec with 200 µL of 80% Ethanol.
Discard ethanol and aspirate remainder with a small 10 µL tip.
Air dry for ~5 min, until beads are dry. Beads will change color slightly when dry.
Add 30 µL of water to resuspend the beads.
Remove from the magnetic stand. Mix well by pipetting or vortexing and incubate for 2 min at room temperature.
Place Magnetic Separator Stand for 1 min. Collect supernatants in new PCR tubes. These are Purified Amplified Samples.


Measure DNA concentrations of Amplified Samples with Qubit HS dsDNA assay
This should be done according to the manufacturer's instructions, found here: https://assets.fishersci.com/TFS-Assets/LSG/manuals/MAN0017455_Qubit_1X_dsDNA_HS_Assay_Kit_UG.pdf. 

1. Measure concentrations using Qubit Fluorometer. 
2. Record concentrations of each Purified Amplified Sample.


Perform Nextera tagmentation
Reagents in this section are from the Nextera XT Library Preparation Kit (Illumina cat. no. FC-131-1096). Tagmentation will fragment the amplified PCR products into shorter sequences, and the length of the reaction time determines how much fragmentation occurs. Therefore, it is very important to follow the times strictly. 
   
In a clean set of PCR tubes, aliquot 7 µL of Neutralize Tagment Buffer (NT). This will be used to stop the tagmentation and prevent over-tagmentation. One tube per sample/well is required. Set aside, next to the thermocycler.
To a new PCR tube for each Purified Amplified Sample (on ice):
        a. Add 10 µL of tagment DNA buffer (TD)
        b. Add 2 ng of purified Purified Amplified Samples and add water up to 5uL. Calculated sample and water volume to add based on the Qubit concentrations from  previous step.
        c. Add 5 µL of Amplicon Tagment Mix (ATM) to lid of tube. This contains the enzyme.
  3. Briefly spin down tubes, vortex, and spin down.
  4. Place into PCR machine and incubate at 55°C for EXACTLY 5 minutes.
  5. Immediately stop reactions with 5 µL of Neutralize Tagment buffer (NT) (use a multichannel to mix). 
  6. Vortex and spin to ensure full mixing. Hold Tagmented Samples on ice.


Sample indexing PCR with unique i5 and i7 primer pairs
To pool Tagmented Samples from each well/replicate for sequencing, each sample is assigned a unique pair of i7 and i5 primer sequences. The primer pairings should be decided and documented. Each Tagmented Sample will be amplified briefly in this step to attached the sample-specific i5 and i7 sequences.

1. Assign and record unique index primer pairs to each sample. For example:
   Samplei5 primeri7 primerSample 1 (Well 1)S502.GATENext.N701Sample 2 (Well 2)S503.GATENext.N702Sample 3 (Well 3)S505.GATENext.N703Sample 4 (Well 4)S506.GATENext.N704
 
 2. To each Tagmented Sample tube from the previous tagmentation step:
        a. Add 6.5 µL water
        b. Add 1.75 µL of standard Nextera (i7: Next.N70X) primer (from 10 uM stock)
        c. Add 1.75 µL of custom Nextera (GATE, i5: S50X.GATE) primer (from 10 uM stock)
        d. Add 15 µL PCR mix (NPM PCR master mix)
 3. Vortex and spin down reactions.
 4. Place in thermocycler and start Indexing PCR Program.
                Indexing PCR Program
                    72°C for 3 minutes
                    95°C for 15 seconds
                    12 cycles of:
                          95°C for 15 seconds
                          55°C for 15 seconds
                          72°C for 40 seconds
                    72°C for 1 minute
                    Hold at 10°C
5. The resulting tubes are the Indexed Samples.


Purify indexed samples with Ampure Beads
Make 50 mL of 80% Ethanol, diluted in UltraPure water.
For each well, combine 50 µL of Indexed Sample and mix with 45 µL Ampure XP Beads in a new PCR tube. 
Mix well by pipetting (incubate at room temperature for 5 min). DO NOT overextend this step.
Place on Magnetic Separator Stand for 2 min.
Discard supernatant. Wash 3 x 30 sec with 200 µL of 80% Ethanol.
Discard ethanol and aspirate remainder with a small 10 µL tip.
Air dry for ~5 min, until beads are dry. Beads will change color slightly when dry.
Add 30 µL of water to resuspend the beads.
Remove from the magnetic stand. Mix well by pipetting or vortexing and incubate for 2 min at room temperature.
Place on Magnetic Separator Stand for 1 min. 
Collect supernatants in new tubes. These are the Purified Indexed Samples.


Measure DNA concentrations and length of Indexed Samples
1. Measure concentrations using Qubit HS dsDNA assay. This should be done according to the manufacturer's instructions, found here: https://assets.fishersci.com/TFS-Assets/LSG/manuals/MAN0017455_Qubit_1X_dsDNA_HS_Assay_Kit_UG.pdf. 
2. Record concentrations of each Purified Indexed Sample.
3. Run 2 uL of each Purified Indexed Sample on a 1% agarose gel.
 
SECTION: Tissue Sectioning and Reverse Transcription
5. Recommended to section onto a coverslip
For Light-Seq, it is critical that light can be focused onto the cells of interest. Thus, there should be minimal disruption of the light-path from the microscope to the sample during barcoding. This is very important for labelling isolated cells within a tightly packed tissue, where barcoding boundaries must be precise. In the publication, we sectioned retinas into chambers on glass coverslips coated with poly-D-lysine to promote tissue adhesion (Ibidi, #80826). We recommend sectioning onto coverslips if possible, and either using the Ibidi chambers or a chamber that sticks onto the coverslips (GraceBio Labs SecureSeal™ Hybridization Chambers https://gracebio.com/product/secureseal-hybridization-chambers-621102/). These come in a variety of sizes and allow multiple tissue sections to be treated separately on the same coverslip. For these, it is important to cover the holes during extended incubations to prevent evaporation (e.g. with a coverslip or a sticker).

Some tissues do not adhere well to coverslips. If problems occur with tissue adhesion, we recommend coating the coverslip prior to sectioning with a poly-D-lysine solution or some other polymer, baking the sample after sectioning, or sectioning onto SuperFrost Plus slides.


For sections on thick microscope slides
It is possible to perform barcoding on tissue sections that are on SuperFrost Plus Adhesion Slides (Thermo Scientific, #J1800AMNZ). We have found that SuperFrost Plus slides promote better tissue adhesion than coated coverslips. In this case, we recommend using the Grace Bio-Labs SecureSeal Chambers for easy fluid exchanges and to prevent evaporation. However, please note that if  barcoding is performed through a thick glass slide then there will be refraction of light through the glass, which can create imprecise barcoding. This is not an issue for barcoding of large regions where the boundaries need not be precise, but for boundary cases where precision of <10 um in the XY-dimension is critical, we do not recommend barcoding through thick slides. 
SECTION: Tissue Sectioning and Reverse Transcription
5.1. Cryosectioning and Reverse Transcription
Sectioning for Light-Seq is ideally performed on a coverslip, to promote optimal light delivery for photocrosslinking of DNA barcodes. In situ reverse transcription (RT) is performed to create cDNA copies of all RNA within the fixed tissue sections and is done using a thermocycler for slides, rather than tubes. We recommend the Eppendorf® Mastercycler® nexus Flat Thermal Cyclers (VWR Cat No. 71003-568).

 
Reagents for Sectioning  
   ReagentsSupplierCat. No.µ-Slide 8 Well, Poly-L-Lysine coatedibidi80826ORGraceBio Labs SecureSeal Hybridization ChambersGraceBio Labs621102LabScientific Rectangular Coverslips, 24 x 50 mm, #1.5, 60 packFisher ScientificLabScientific 7816TWEEN® 20Sigma AldrichP9416- 50MLPBS - Phosphate-Buffered Saline (10X) pH 7.4, RNase-freeInvitrogenAM962520X Borate BufferThermo Fisher28341Poly-D-lysine hydrobromideMillipore SigmaP6407-5MGUltraPure WaterInvitrogen10977023


Reagents for Reverse Transcription
        Reagents  Supplier  Cat. No.          Thermo Scientific™ Maxima H Minus    Reverse Transcriptase (200 U/μL)    (includes 5X buffer)  Thermo Scientific  FEREP0753    Triton-X-100  Sigma    Aldrich  T8787-    50ML    5   M NaCl  Invitrogen  AM9760G    TWEEN® 20  Sigma Aldrich  P9416-    50ML    PBS  Invitrogen  AM9625    Formamide        UltraPure   Water  Invitrogen  10977023    Deoxynucleotide   (dNTP) Solution Mix    - 8 μmol at 10mM each  NEB  N0447S    RNaseOUT™   Recombinant    Ribonuclease Inhibitor  Invitrogen  10777019  

Reverse Transcription DNA Oligos 
     NameDescriptionSequenceSupplierPurificationRT.5N.3GRT PrimerTTTACACGATTGAGTTATNNNNNGGGIDTHPLC
 

Section and perform in situ reverse transcription
Prepare solutions:
1X PBS (40 mL):
4 mL 10X PBS
36 mL UltraPure water

0.1% PBS-Tw (30 mL):
30 mL 1X PBS 
30 μL Tween-20

PDL Solution
0.3 mg/mL poly-D-lysine dissolved in 2X Borate Buffer
    Note: Store in aliquots at -20C. Do not freeze-thaw.

Stringent Wash (0.1% PBS-Tw + 60% formamide):
3 mL 10X PBS
18 mL 100% formamide
9 mL UltraPure H2O
30 μL Tween-20
Note: Store at 4C for use later during barcoding.

High-Salt Wash (1X PBS + 1 M NaCl + 0.1% Tween- 20):
4 mL 10X PBS
8 mL 5 M NaCl
28 mL UltraPure H2O
40 μL Tween-20
Note: Store at room temperature for use later during barcoding.

10% Triton X-100 (100 μl):
10 μL Triton X-100 
90 μL UltraPure water
    Note: Mix very well by vortexing. Recommended to make this in a 2 mL tube for better mixing. 

2. Coat coverslip with PDL.
- For Ibidi Chambers: Pipette PDL Solution onto the coverslip and let sit for 2 hours or overnight at 4C. Then remove PDL Solution, allow slide to dry completely, and wash once with UltraPure water.
- For SuperFrost slides or coverslips: Pipette 500 μL of PDL Solution onto the slide and let it sit for 1min. Then remove all liquid, allow slide to dry completely, and wash once with UltraPure water.

3. Trace locations of Grace BioLabs Chambers on the back of the coverslip to guide section placement.
4. Prepare Reverse Transcription Master Mix (RTMM) on ice, leaving out enzymes (Maxima RT H minus and RNAse Out). Enzymes will be added directly before RT.

Reverse Transcription Master Mix (RTMM)
 

    5. Section tissue on a cryostat, thickness of 5-18 μm.
    6. Once all sections are on the coverslip, bring to room temperature. 
    7. Immediately spin the coverslip in a plate-centrifuge at 600g for 3 min to promote tissue adhesion.
    8. Allow sections to dry completely at room temperature (~10min, time depends on section thickness). 
    9. Wash sections 3 x 1 min with 1X PBS to fully remove cryoprotectant.
    10. Allow coverslip to dry completely between sections (use aspirator or Kimwipe). 
    11. Adhere Grace BioLabs Chambers to coverslip.
    12. Wash sections 3 x 1 min with 0.1% PBSTw.
    13. Add enzymes (Maxima RT H minus and RNAse Out) to RTMM and mix well.
    14. Remove 0.1% PBSTw and add 50 ul of RTMM to each well. 
    15. Place slide into Eppendorf® Mastercycler® nexus Flat and run RT program (2h 20 min). Place a second coverslip over the chambers to prevent evaporation.

            Reverse Transcription Thermocycler Program
            12-cycle ramp program (Lid: 60°C):
            Phase 1: 
                 22°C      30 mins 
            Phase 2 (12 cycles): 
                        8°C        30 s 
                        15°C      30 s 
                        25°C      30 s 
                        30°C      1 min 
                        37°C      1 min 
                        42°C     2 min  
            Phase 3:    
                42°C      30 min  
                 4°C        Forever
 
    16. Wash each well 3 x 5 min in Stringent Wash.
    17. Wash each well 2 x 2 min in High Salt Wash.
    18. Wash each well 2 x 2 min in 0.1% PBS-Tw.
    19. Fill each well with 50 ul of 0.1% PBS-Tw.

Optional Pause Point: Store at 4C overnight before A-tailing.

NOTE: Sectioning for the publication was done immediately before reverse transcription. It is possible, however, to section and immediately freeze the sections at -80C. If using frozen sections, start from step (8) above, and allow sections to dry completely after thawing. To preserve RNA quality, the time between section thawing and RT should be minimal.
SECTION: Protocol Overview
1. This protocol is for performing Light-Seq in tissue samples, as published in Nature Methods. The typical experiment can be performed by a single scientist in one work week, with natural pause points.

Day 0: 
Tissue Dissection & Fixation (~2 hours)

Day 1:
Tissue Sectioning (1 hour)
In Situ Reverse Transcription (2.5 hours)
A-tailing (1 hour) 
Antibody Stain (optional, 1-3 hours or overnight)
  

Day 2: 
Light-Directed Barcoding (~4 hours for 3 ROIs and 3-6 replicates)
  
Day 3: 
cDNA Displacement (1 hour)
Cross-Junction Synthesis (2 hours)
  

Day 4: 
qPCR and PCR Amplification (5 hours)
  

Day 5: 
Library Preparation for Next-Generation Sequencing (3 hours)
SECTION: qPCR Amplification
10. Cross-Junction Synthesis libraries of barcoded cDNAs are now amplified and prepared for sequencing. We use the Kapa Hifi HotStart polymerase. We recommend performing qPCR with 30 cycles on a small subset of each sample (5 uL sample in a 10 uL PCR), to identify the appropriate cycle number for optimal amplification. 

qPCR Primers
     NameDescriptionSequenceSupplierPurificationGATEPCR Primer 1GGAGTTGGAGTGAGTGGATGAGTGATGIDTHPLCGATCPCR Primer 2GAGAATGTGAGTGAAGATGTATGGTGAIDTHPLC
 
Reagents for qPCR 
   ReagentsSupplierCat. No.SYBR™ Green I Nucleic Acid Gel Stain - 10,000X concentrate in DMSOInvitrogenS7563HiFi HotStart DNA Polymerase, KapaBiosystemsRocheKK2502UltraPure WaterInvitrogen10977023

qPCR to Determine Amplification Cycle Number
1. Vortex  CJS Samples.
2. Dilute Sybr Green I dye in water to make 5x solution (e.g. dilute 10,000X stock twice, 1:100 in water, then 1:20 in water).
3. Create PCR master mix:
 
 

4. Quickly vortex and spin down master mix, then add 5 uL master mix into each tube.
5. Add 5 uL of appropriate sample from Cross-Junction Synthesis reaction to each tube.
6. Quickly vortex and spin down reactions.
7. Place into qPCR machine for 30 cycles and run the qPCR Machine Protocol.
            qPCR Machine Protocol
                98C for 3 minutes
                30 cycles of:
                    98C for 20 seconds
                    60C for 30 seconds
                    72C for 2 minutes
                    Plate read
                72C for 5 minutes
                Melting curve analysis
                Hold at 4C
8. Check amplification graph to choose appropriate cycle number.
 

SECTION: Tissue Dissection and Fixation
4. Tissue Fixation and Embedding Reagents 
   ReagentsSupplierCat. No.Triton-X-100Sigma AldrichT8787- 50MLPierce™ 16% Formaldehyde (w/v), Methanol-freeThermo Scientific28908PBS - Phosphate-Buffered Saline (10X) pH 7.4, RNase-freeInvitrogenAM9625Tissue-Tek® O.C.T. CompoundSakura4583Sucrose, Molecular Biology GradeThermo ScientificJ65148.A1

Retina dissection, fixation and embedding
Prepare solutions:
4% PFA, 0.25% TritonX-100 in 1X PBS (40 ml):
10 mL 16% PFA ampule
4 mL 10X PBS (molecular-grade)
26 mL nuclease-free water
100 μl Triton-X-100
    Note: Buffer should be prepared in fume hood as formaldehyde is toxic. Please prepare this fresh and do not freeze.

30% sucrose in 1X PBS:
15 mg sucrose
in final volume of 50 mL 1X PBS
    Note: Filter-sterilize using 0.4 μm filter and store at 4C.

7% sucrose in 1X PBS:
3.5 mg sucrose
in final volume of 50 mL 1X PBS
    Note: Filter-sterilize using 0.4 μm filter and store at 4C.

Cryoprotectant Solution:
50 mL of 1:1 mixture of [30% sucrose in 1X PBS] : [Tissue-Tek O.C.T. Compound]
    Note: This mixture will take time to mix thoroughly. Vortexing will help, but be sure to spin it down after to remove bubbles before applying to the tissue. 

2. Dissect mouse retinas in 1X PBS at room temperature. 
3. Immediately transfer retinas to 4% PFA, 0.25% TritonX-100 in 1X PBS for 25 mins at room temperature, rocking.
4. Wash 3 x 5 min in 1X PBS.
5. Transfer retinas to 7% sucrose in 1X PBS for 10 min, rocking.
6. Transfer retinas to Cryoprotectant Solution for 30 min rocking, to equilibrate the tissue in cryoprotectant before freezing. 
7. Transfer retinas to cryomolds and freeze in Cryoprotectant Solution.
8. Store frozen retinas at -80C for up to 6 months (possibly longer, but not tested). 

SECTION: Tissue Dissection and Fixation
3. Determining tissue-specific fixation/preservation protocol
The protocol outlined here is for cryo-sectioned mouse retinas. Different tissues require different optimizations for fixation and sample preparation. We highly recommend using standard tissue-specific protocols that are compatible with RNA detection (FISH, for example) as a starting point for Light-Seq.

Times for fixation, washing, and incubation will vary for different sample types. In general, we find that over-fixation can greatly impair Light-Seq barcoding efficiency and in some cases, antigen retrieval protocols are required (e.g. for FFPE). It is recommended to minimally but sufficiently fix your tissue and to freeze immediately to preserve RNA quality. As a starting point, we recommend starting with standard tissue-specific protocols that are compatible with in situ RNA FISH. 

For users who have not performed FISH in their samples before, we recommend testing the RNA quality in your tissue before proceeding with Light-Seq. This could be done using any number of RNA-detection methods, including HCR, RNA-scope, etc. We expect that tissue preparation parameters that work well for these in situ RNA detection methods should also work well for Light-Seq.
SECTION: Light-Directed Barcoding of cDNAs
7. Choosing an Optical System
Before starting, ensure that your optical system is set up and calibrated properly, so that UV light can be focused on cells/regions of interest (ROIs). 
SECTION: PCR Amplification
None. 
SECTION: Sequencing
13. This library preparation is compatible with standard Illumina next-generation sequencing. Note that custom Read 1 and i5 index primers are required and are listed in the table below.

DNA Oligos for Sequencing 
    DescriptionSequenceSupplierPurificationCustom Read 1 Primer -required for sequencing ofamplicons.CGCCGGAGTTGGAGTGAGTGGATGAGTGATGIDTHPLCCustom i5 index primer -required for some Illuminasequencers (see caption).CATCACTCATCCACTCACTCCAACTCCGGCGIDTHPLC
 
We have found adding 60% above the standard concentration of each custom primer works well on many of the Illumina sequencers and would recommend this as a starting point.

We have had good success with elevated custom primer concentrations on Illumina MiSeq and NextSeq machines. The standard custom primer concentrations worked for HiSeq. For NovaSeq, we haven't seen as good efficiency even with the elevated custom primer concentrations, particularly for the custom i5 index sequencing but hope to have updated recommendations soon. For now, we recommend having unique i7 index sequences for each sample to aid in sequence de-multiplexing, and that you check back here periodically or reach out for updated sequencer-specific recommendations.
SECTION: Post-Sequencing Tissue Staining
14. Tissue can stored for 2+ weeks in 1X PBS and can be stained after sequencing. You should cover the sample appropriately to ensure that it does not dry out. We recommend also using a hybridization chamber, and replensihing the liquid in the wells every few days. While some antigens might be disrupted by the protocol, we have detected proteins via immunofluorescence with success in addition to other stains.