TITLE: OMS Atlas OCT Spatial Mapping
AUTHORS: Brett Johnson, Danielle Galipeau, Todd Camp, George Thomas

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes the procedure by which the OMS Atlas serially sections an OCT block, prepares the resulting slides and samples, and then distributes the specimens for downstream analysis.</div></div>

[STEPS]
?. [Preparation]
Verify the identity of the OCT block to be cut against written request for sectioning.
?. [Preparation]
Remove OCT block from  freezer and acclimate to cryostat ( ) for minimum of .
-80 °C
-20 °C
?. [Preparation]
Label all slides and cryotubes with a unique BEMS ID and Part#, corresponding to the written request and OCT spatial map (below).  ABCDE1Part#DescriptionThicknessAssayRecipient21Tanner slide5µmCyclic ImmunofluorescenceOHSU, Koei Chin32Tanner slide5µmH&EOHSU, HSR43Tanner slide5µmCyclic Immunofluorescence (Tumor Panel)HMS, Alyce Chen54Tanner slide5µm (Set Cryostat at 12µm)Cyclic Immunofluorescence (Tumor Panel)HMS, Alyce Chen65Cryotube7µmSingle Cell DNA SequencingMD Anderson, Nick Navin76PEN membrane slide12µmTopographic Single Cell SequencingMD Anderson, Nick Navin87PEN membrane slide12µm (Set Cryostat at 40µm)Topographic Single Cell SequencingMD Anderson, Nick Navin98Cryotube40µm (2 sections)Single Cell DNA SequencingMD Anderson, Nick Navin109Remainder of OCT blockNASingle Cell Indexing ATAC SequencingOHSU, Andrew Adey
ABCDE1Part#DescriptionThicknessAssayRecipient21Tanner slide5µmCyclic ImmunofluorescenceOHSU, Koei Chin32Tanner slide5µmH&EOHSU, HSR43Tanner slide5µmCyclic Immunofluorescence (Tumor Panel)HMS, Alyce Chen54Tanner slide5µm (Set Cryostat at 12µm)Cyclic Immunofluorescence (Tumor Panel)HMS, Alyce Chen65Cryotube7µmSingle Cell DNA SequencingMD Anderson, Nick Navin76PEN membrane slide12µmTopographic Single Cell SequencingMD Anderson, Nick Navin87PEN membrane slide12µm (Set Cryostat at 40µm)Topographic Single Cell SequencingMD Anderson, Nick Navin98Cryotube40µm (2 sections)Single Cell DNA SequencingMD Anderson, Nick Navin109Remainder of OCT blockNASingle Cell Indexing ATAC SequencingOHSU, Andrew Adey
?. [Preparation]
Prepare PEN membrane slides by exposing close (~15-20cm) to a UV source for  .
?. [Sectioning]
Affix OCT block to cryostat chuck.
?. [Sectioning]
Orient and face block to get adequate amount of core.Note: Avoid excessive facing to reduce tissue loss.
?. [Sectioning]
Set cryostat to 5 micron sections.Note: All sections cut from here on should be sequential. The serial order, adjacency, and consistent orientation of the sections are all important factors. Please note any deviations from the protocol.
?. [Sectioning]
Cut first four sections at 5 microns (Part#1-4) and affix onto appropriately labeled Tanner slide according to OCT spatial map (step #3 above).
?. [Sectioning]
Change section thickness to 12 microns.
?. [Sectioning]
Cut one section (Part#5) and place in a cryotube.Note: This is an intermediate section generated when the Cryostat is switching thicknesses. The actual thickness of this section should be about 7µm.
?. [Sectioning]
Cut two sections (Part#6, 7) and place on appropriate membrane slides.
?. [Sectioning]
Change section thickness to 40 microns.
?. [Sectioning]
Cut 2 sections (Part#8) and place both sections in a single cryotube.
?. [Sectioning]
Place all slides, both cryotubes, and remaining OCT block in  freezer.Note: No slides are to be fixed under this protocol.
-80 °C
?. [Processing]
Perform hematoxylin and eosin (H&E) staining on slide labeled Part#2 (see OCT spatial map in step #3 above).
?. [Processing]
Deliver unstained slides (Part#1, 3, 4, 6, 7), cryotubes (Part#5, 8), and remainder OCT block (Part#9) to BioLibrary for distribution.Note: Keep samples frozen at all times. Store at . Transfer/ship on dry ice.
-80 °C