TITLE: Spatial Transcriptomics Protocol
AUTHORS: Michael  Eadon, Ricardo  Melo Ferreira, Ying-Hua Cheng, Tarek  Ashkar

[DESCRIPTION]
The tissue in OCT undergoes cryosectioning, affixment to the cDNA capture slide, H+E staining, 20x Keyence imaging, tissue permeabilization, RNA capture, and cDNA synthesis. Data is analyzed in Loupe browser and in R prior to uploading to the KPMP Data Lake.

[GUIDELINES]
Protocol Quality Control Metrics
A number of our QC metrics have been recommended by 10X Genomics. We will adhere to the recommended commercial standards, specifically an input RIN of greater than 7. Based on our preliminary data, it may be possible to interrogate samples of reduced input quality and still obtain adequate sample output quality for analysis. If bulk RIN is higher than 3.8 we intend to procced with interrogation, flagging the sample if it meets output QC metrics established by 10X genomics. The QC metrics include:
Bulk RNA quality, Ideal RIN should be greater than 7, according to 10X genomics. Samples with a RIN between 3.8 and 6.9 will be interrogated and flagged if meets downstream QC metrics. Please see the preliminary data below justifying a reduced RIN threshold. Samples with a Bulk RIN less than 3.8 will not be interrogated (No-Go criteria). We chose 3.8 because two samples with a RIN of 3.8 met the 10x genomics defined QC output metrics. There is some trade-off in using samples with lower input QC metrics (i.e. a RIN between 3.8 and 6.9), but given that 1) we demonstrate these samples meet the output QC metrics defined by 10x and 2) the overall value of this technology is important, we feel that the trade-off is acceptable. This RIN threshold could be re-assessed after processing of additional biopsies (once approved).
Tissue affixment without folds or overlap – if folded, wipe and re-apply
Keyence image visual inspection – if inadequate resolution, reimage single specimen (6 minutes). The visual inspection will be performed by two individuals including the core microscopist and KPMP biologist. Minimum standards include the ability to identify the following four features: sharp nuclei, glomeruli, proximal tubules, and distal tubules.
Post-permeabilization RNA quality and quantity – sigmoid curve shape, sufficient RNA before cycle 18-20, per 10X Genomics published QC metrics.
cDNA quality – check that it is not fragmented. Satisfactory values are usually above 500, but this is not a hard Go, No-Go criteria.
Sequencing Q30 – better than 90%, per 10X Genomics published QC metrics.
Transcript mapping – majority of reads (more than 50%) must map to spots with tissue overlying, per 10X Genomics published QC metrics. Sample will be flagged if not meeting this threshold.
Reads mapped confidently to Exons should be above 30%, per 10X Genomics published QC metrics. Sample will be flagged if not meeting this threshold.
Picture of alignment process is taken to ensure fiducials correspond – correct informatically if misaligned. Fiducials are defined as spots around the periphery of the capture area to allow image alignment.
Expected differential expression of marker genes in spot clusters.
Sequencing saturation will be tracked to gauge the appropriate depth of sequencing and assess for drift. However, this is not a quantitative output metric.

[STEPS]
SECTION: Cryosectioning Procedure
1. Specimens should be handled wearing clean gloves treated with RNAse Away or with a clean RNAse Away treated forceps.
 

SECTION: Cryosectioning Procedure
2. Cool Cryostat to -22 °C. Clean the work surfaces with RNAse away and install a new cutting blade.

SECTION: Cryosectioning Procedure
3. Place a clean small slide box inside the cryostat chamber to store slides containing freshly cut tissue.
SECTION: Cryosectioning Procedure
4. Clean and treat the tissue holder will RNAse away and put it in the cryostat chamber . Adhere the OCT specimen to a tissue holder and allow it to equilibrate for a few minutes to reach the chamber temperature and strengthen the adhesion between the OCT block and the holder. This process can be aided via use of a heat extractor. The tissue holder was cleaned and treated with RNAse Away and cooled down.
SECTION: Cryosectioning Procedure
5. In parallel, allow the cDNA capture slide (Visium product code #1000184) to equilibrate at -20 °C.

SECTION: Cryosectioning Procedure
6. Place the specimen in the cryostat with the tissue holder and cut at    thickness (1 section). 
 


SECTION: Cryosectioning Procedure
7. Try to avoid overlap with fiducials surrounding the capture area. If that is not possible, this can be adjusted informatically after sequencing if there is overlap. A minimum of 2 corners must be clear of tissue.
SECTION: Cryosectioning Procedure
8. Store completed slides at -80 °C  in sealed 50mL RNAse free conical centrifuge tubes (Cat#91050). Use slides within 48 hours of cryosectioning.

SECTION: Cryosectioning Procedure
9. Process specimens in batches of four because there are 4 capture areas per slide.
SECTION: Cryosectioning Procedure
10. If tissue is folded or misplaced, the slide can be reset using Visium Spatial Slide Reset Protocol (CG000332 • Rev B) and reuse.
SECTION: Fixation and Hematoxylin and Eosin staining
11. Use the Visium library preparation slide with affixed OCT sections and perform H+E staining.
SECTION: Fixation and Hematoxylin and Eosin staining
12. Chill 40 mL Methanol in a 50-ml centrifuge tube to -20 °C.

SECTION: Fixation and Hematoxylin and Eosin staining
13. Place a Thermocycler Adaptor on thermal cycler set at 37 °C and equilibrate for 5 min. Heating the thermal cycler lid is not required.

SECTION: Fixation and Hematoxylin and Eosin staining
14. Place slide on the Thermocyler Adaptor with the active surface facing up and incubate 1 min at 37 °C. DO NOT close the thermal cycler lid, as the thermal cycler lid is normally heated to a higher temperature and it may touch the tissue sections when closed.

SECTION: Fixation and Hematoxylin and Eosin staining
15. Completely immerse the slide in the prechilled methanol. Secure the tube cap. Incubate upright for30 min at -20 °C.

SECTION: Fixation and Hematoxylin and Eosin staining
16. Remove slide from methanol and wipe excess liquid from the back of the slide without touching the tissue sections.
SECTION: Fixation and Hematoxylin and Eosin staining
17. Add 500 µL isopropanol to uniformly cover all tissue sections on the slide. Incubate 1 min at -20 Room temperature.

SECTION: Fixation and Hematoxylin and Eosin staining
18. Drain reagent and air dry the slide for 6 min.

SECTION: Fixation and Hematoxylin and Eosin staining
19. Add 1 mL Hematoxylin to uniformly cover all tissue sections on the slide. Incubate 7 min at -20 Room temperature.

SECTION: Fixation and Hematoxylin and Eosin staining
20. Discard reagent and rinse slide sequentially in RNase free water. Wipe away excess liquid.
SECTION: Fixation and Hematoxylin and Eosin staining
21. Add 1 mL Bluing Buffer to uniformly cover all tissue sections. Incubate 2 min  at -20 Room temperature.

SECTION: Fixation and Hematoxylin and Eosin staining
22. Discard reagent and rinse slide sequentially in RNase free water. Wipe away excess liquid.
SECTION: Fixation and Hematoxylin and Eosin staining
23. Add 1 mL Eosin Mix to uniformly cover all tissue sections. Incubate 1 min at -20 Room temperature.

SECTION: Fixation and Hematoxylin and Eosin staining
24. Discard reagent and rinse slide sequentially in RNase free water. Wipe away excess liquid.
SECTION: Fixation and Hematoxylin and Eosin staining
25. Allow slide to air dry for 6 min then incubate the slide on the Thermocycler Adaptor with the thermal cycler lid open for 5 min at 37 °C prior to imaging

SECTION: Imaging
26. After the specimen is fixed and stained with H+E reagents it is then imaged on a Keyence BZ-X810 scanning microscope at 10x or 20x resolution in the IU O’Brien Center for Microscopy.
 

SECTION: Imaging
27. Brightfield images of stained sections in the fiducial frames are collected as mosaics of 20x fields using a Keyence BZ-X810 microscope equipped with a Nikon 10X CFI Plan Fluor objective.
SECTION: Imaging
28. Including set-up time, 24 min was required to image 4 sections. Image quality is assessed by visual inspection prior to RNA isolation and a given section can be re-imaged if necessary (~6 min needed).

SECTION: Imaging
29. Mosaics are stitched.
SECTION: cDNA capture and sequencing
30. Bulk RNA quality is assessed on an adjacent section with an Agilent bioanalyzer prior to sectioning the tissue for spatial transcriptomics. Ideal RIN should be above 7, although some specimens may be able to yield adequate data with a RIN as low as 3.8.
SECTION: cDNA capture and sequencing
31. Permeabilize stained tissue sections for 12 min, mRNA was released to bind oligonucleotides on the capture areas with a test for RNA quality/quantity after permeabilization and isolation using real time PCR amplification from the Visium library prep slide.

SECTION: cDNA capture and sequencing
32. cDNA synthesis - reverse transcription, second strand synthesis, denaturation, cDNA amplification, SPRIselect cDNA cleanup. cDNA quality is assessed by Agilent bioanalyzer.
SECTION: cDNA capture and sequencing
33. Prepare and sequence the cDNA libraries on an Illumina NovaSeq 6000 with 28bp+120bp paired-end sequencing mode.
SECTION: cDNA capture and sequencing
34. Using a Loupe Browser, map expression data to each “spot” and visualize overlying the histologic image