TITLE: Preparation of Adult Human Kidney Tissue for Single Nucleus RNA-seq and Other Multiomics Studies
AUTHORS: sanjay jain, Masato Hoshi, Amanda Knoten, Diane Salamon

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Critical to generating robust single cell or tissue gene expression data is ensuring that the source samples are procured and processed in a way that minimizes artifacts. This is especially critical for generating single cell/nucleus gene expression data from adult human tissue samples that are difficult to dissociate and might require prolonged manipulations that will introduce stress response signatures. We describe methods to: minimize these unwanted processing effects to enable interrogation of limited tissues, such as biopsies; allow histological validation and quality assurance and control of the samples; ensure compatibility with both single cell assays and orthogonal technologies. This involves preparing fresh frozen O.C.T. embedded tissue cryoblocks to collect thick sections for gene expression studies and adjacent sections for QA/QC, bulk RNAseq, DNA preparation or multiplex in situ RNA hybridization.   The protocol for embedding limited amount of tissue for cryosections and susequent OMICS studies should be applicable to several solid organs and biopsies.
 
 
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[STEPS]
?. [FrozenTissue embedding and OCT Block Preparation]
Label one cryomold with specimen number and place it on powdered dry ice. This will be the cassette in which tissue will be embedded.
?. [FrozenTissue embedding and OCT Block Preparation]
Take the second cryomold and fill it with O.C.T.
Tissue will be bathed in it before transferring to the cryomold in step 1.
?. [FrozenTissue embedding and OCT Block Preparation]
Rapidly dissect the tissue, dab away excess blood using a pad or gauze. Cut desired size section that will fit easily in the cryomold chosen and briefly bathe the tissue in O.C.T. in the cassette in step 2, gently swishing around with a clean instrument  (Figure 1A).
Keep separate forceps to handle tissue during dissection from the one used for bathing as the O.C.T. will stick to the forceps and can make dissection difficult. Handle the tissue gently, for a long biopsy one could also use a pipette tip held in hand to bathe the tissue and lift to transfer without squeezing it.
?. [FrozenTissue embedding and OCT Block Preparation]
QA/QC: Using a smartphone camera, an image of the tissue can be captured while the tissue is bathing that can be used for gross identification of cortex and documentation.
?. [FrozenTissue embedding and OCT Block Preparation]
Transfer the tissue into the pre-labeled and chilled cryomold (step 1), orient the tissue as desired and pour O.C.T. till it completely covers the tissue (Figure 1B, C).
Avoid bubbles; can move the bubbles out of the way with forceps or pipette tip.
?. [FrozenTissue embedding and OCT Block Preparation]
Keep the cassette on powdered dry ice for freezing. Keep the dry ice bucket covered to keep a cold environment. Avoid flash freezing as that could result in tissue fracturing and thus loss of integrity.
?. [FrozenTissue embedding and OCT Block Preparation]
Transfer the frozen block into a pre-chilled ziplock (on dry ice), seal it taking as much air out as possible and keeping the block chilled, and store it in a freezer box at  until ready to section or ship overnight packed in dry ice shipping container. To avoid damaging the block during transit, place it securely in a pre-chilled freezer box and pack in a Styrofoam container with ample dry ice.
-80 °C
?. [Sectioning]
Acclimate O.C.T. blocks in cryostat for about  (check cryostat temperature, usually kept at  to ) .
-20 °C
-25 °C
?. [Sectioning]
Prepare tubes, slides, and containers before cutting and as needed.
?. [Sectioning]
At  spray all instruments (forceps/paintbrushes) with RNaseZAP.
0 Room temperature
?. [Sectioning]
Add O.C.T. to a pre-chilled metal chuck kept in the cryostat and while freezing place the O.C.T. block with the tissue (tissue facing up) on the chuck. In a few minutes the O.C.T. block with the tissue will be attached to the metal chuck.
?. [Sectioning]
Mount block with the tissue on the cryostat stage (tissue facing the user).
Caution: Be careful when inserting or changing or cutting with cryostat blade and use gloves while cutting. All tissue should be handled keeping universal precautions in mind and consider them biohazardous. In the event of injury contact your local safety office
?. [Sectioning]
Sections are collected as described below for various processing conditions (see table as an example, the thickness of sections for histology can be at 3um.  The preservative solution can be different depending on reproducibility and number of orthagonal technologies to be tested.
?. [Sectioning]
Thick section rolls (35-40 μm) (use for RNA or protein studies) (series of tubes with tissue curls allows for reproducibility testing and comparing different processing methods – QA/QC) are collected in microfuge tubes (Figure 3).
Examples:Dry Ice, no solution: 7 rolled sections (total 280μm) collected in chilled 1.7ml  centrifuge tube. Keep chilled at all times so OCT doesn’t melt. Process immediately. Used for: RNA or protein assays - e.g. snRNA-seq or single cell chromatin accessibility. RNALater: 7 rolled sections are collected in centrifuge tube containing 1mL RNAlater and kept chilled on ice (RNA - snRNA-seq or total RNA). At this stage, the RNAlater containing tubes can be shipped overnight (packed with ice packs) to another site or stored at -20°C for a few weeks (RNAlater will not freeze). Used for: RNA assays – e.g. snRNA-seq or total RNA isolation. Collected in Other media: as needed
Be careful when handling the tubes, avoid touching the surface with the tissue as it could cause local thawing, better to hold at the lip of the tube or anywhere that is away from the tissue if need be.
?. [Sectioning]
Sections for coverslips are collected as follows (for DART-FISH):
?. [Sectioning]
Keep the prepared coverslips at .
0 Room temperature
?. [Sectioning]
Section tissue at desired thickness (we cut at 10 μM and are in the process of testing 20 μM) and place the coverslip so tissue is in the center. Use forceps to pick up and flip the coverslip so the tissue is now facing upwards. Keep chilled.
Be gentle and avoid pressing too firmly as this will smash the tissue. Gently tilting the coverslip (or slides in case of histology) usually will cause the tissue to lift and adhere due to static generated.
Adjacent tissue sections allows for reproducibility testing and comparing different processing conditions – QA/QC
?. [Sectioning]
Pre-chill the tubes for RNAlater in wet ice. Slides on which sections will be mounted for histology can be at room temperature.
?. [Sectioning]
Sections on slides are collected in 3-5 slide intervals at 5-10 μm, 1 section per slide, for histology/immunofluorescence to validate the tissue being used. Slowly bring slide to the cut section and as sections transfer to the slides, briefly touch the back surface of the slide (opposite to the section) with your finger to facilitate adherence of the tissue section. 
 One slide from each interval will be H&E stained for adequacy, quality, composition and integrity (QA/QC). Keep the remainder in the cryostat and transfer to.
-80 °C
?. [Sectioning]
Place a slide in the cryostat and, once chilled, put a drop of water (~) towards the center of the slide and place coverslip (tissue facing upwards) over the water.Keep the slide flat until the water freezes, attaching the coverslip to the slide. This makes it easier to ship the coverslips.
25 µl
?. [Sectioning]
Place slides with the attached coverslip in pre-chilled slide container for shipment or store at .
80 °C