Systematic Sampling of the Female Reproductive System for Molecular Characterization.

This protocol describes dissociation of snap-frozen tissue in order to isolate nuclei that can be used for downstream analysis. This protocol can also be used on fresh tissue. We used this protocol on snap-frozen cervical (endocervical and ectocervical) tissue and snap-frozen uterine tissue. Unlike the ovary and Fallopian tube, manual dissociation protocols on the cervix and uterine tissue did not yield sufficient quantities of intact nuclei with minimal debris. This protocol is available on protocols.io at https://dx.doi.org/10.17504/protocols.io.yxmvmndx6g3p/v1 ( Fisher et al., 2022k ). Snap-frozen tissue block Nuclei Isolation kit (S2 Genomics, cat. no. 100–060-817) Nuclease-free water Dry ice Protector RNase Inhibitor, 0.6U/μL (Millipore-Sigma, cat. no. 3335399001) Ice Wash buffer (see recipe in Reagents and Solutions ) Trypan blue (Thermo Fisher Scientific, cat. no. T10282 ) Singulator 100 System (S2 Genomics, cat. no. 100–067-764) Disposable weigh boats (VWR, cat. no. 10803–166) Ice bucket (Corning ™ , cat. no. 432122) 20μm cell strainer (Pluriselect, cat. no. 43–50020-03) FACS tubes or 15mL conical tubes (Eppendorf, cat. no. 0030122151) NOTE: Following this protocol, the resulting libraries will be ready for sequencing (for example, with an Illumina NovaSeq tm ). For more details about sequencing the libraries, consult the 10X Genomics “Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Reagent Kits User Guide” at https://www.10xgenomics.com/support/single-cell-multiome-atac-plus-gene-expression/documentation/steps/library-prep/chromium-next-gem-single-cell-multiome-atac-plus-gene-expression-reagent-kits-user-guide . Pre-chill nuclei cartridge in the fridge overnight. TIP: Keep cartridge at 4°C until ready for use. Turn the Singulator 100 on by turning on the main machine, tablet, then cooler chamber (in that order). Place the Nuclei Isolation Reagent (NIR) and Nuclei Storage Reagent (NSR) in the cooler and connect them by attaching the correctly labeled luer lock on the machine. The NIR and NSR can be found in the S2 Genomics Nuclei Isolation Kit. If the waste and water bottles are not yet connected, connect those to the machine using the correctly labeled luer locks. Fill water bottle with nuclease-free water. Press “cool” to begin cooling the chamber. While chamber is cooling, quickly weigh sample (optimal sample weight is 25–100mg). Place sample back on dry ice until ready for use. Set the following parameters for the S2 Genomics “Extended Nuclei Protocol” on the Singulator 100. Auto mince: NO Incubation time: 5 minutes Incubation temperature: COLD Mixing type: TOP Mixing speed: FAST Disruption type: DEFAULT Disruption speed (tissue dependent): Cervix (endocervical and ectocervical) tissue: FASTEST Uterine tissue: FAST Auto mince: NO Incubation time: 5 minutes Incubation temperature: COLD Mixing type: TOP Mixing speed: FAST Disruption type: DEFAULT Disruption speed (tissue dependent): Cervix (endocervical and ectocervical) tissue: FASTEST Uterine tissue: FAST Cervix (endocervical and ectocervical) tissue: FASTEST Uterine tissue: FAST Load nuclei cartridge with sample and 30μL of Protector RNase Inhibitor. Run protocol. TIP: Watch the camera until the NIR has been dispensed. You may need to jiggle the NIR bottle in the cooling chamber to release the tubing from suctioning the side of the NIR bottle – you should only have to do that once. When protocol has finished running, remove the cartridge from the machine and immediately place on ice. Puncture the aluminum foil seal on the cartridge and remove the nuclei suspension. Pellet suspension at 500 × g for 5 minutes at 4°C. Remove and discard supernatant. Resuspend nuclei in 1mL wash buffer and place on ice. TIP: Additional washes may be necessary based on the quality control checks in step 18 (do not exceed 4 washes). Additional clean-ups may also be needed, such as an OptiPrep gradient clean-up as described in “Debris Removal” ( Martelotto, 2021 ). Strain nuclei suspension through a 20μm strainer into a fresh FACS or 15mL conical tube. Using Trypan blue, load nuclei onto a hemocytometer and count. QUALITY CONTROL: Check under microscope for debris. There are specific acceptable levels of blebbing that we are looking for, specifically levels as represented in images A and B from Panel A in 10X Genomics “Nuclei Isolation from Complex Tissues for Single Cell Multiome ATAC + Gene Expression Sequencing demonstration protocol.” (see https://www.10xgenomics.com/support/single-cell-multiome-atac-plus-gene-expression/documentation/steps/sample-prep/nuclei-isolation-from-complex-tissues-for-single-cell-multiome-atac-plus-gene-expression-sequencing ). We’re looking for <5% live cells (ideally no cells), minimal to no clumping, no large debris, minimal to no other debris. TIP: Step 16 above can be repeated 3 more times if a large amount of debris is still present (do not exceed 4 total washes). Process the isolated nuclei with the 10X Genomics permeabilization protocol described in section 1.2 Nuclei Permeabilization of “Nuclei Isolation from Complex Tissues for Single Cell Multiome ATAC + Gene Expression Sequencing demonstration protocol.” (see https://www.10xgenomics.com/support/single-cell-multiome-atac-plus-gene-expression/documentation/steps/sample-prep/nuclei-isolation-from-complex-tissues-for-single-cell-multiome-atac-plus-gene-expression-sequencing ). Protector RNase Inhibitor concentration is 0.6U/μL. Process the permeabilized nuclei with the 10X Genomics Multiomic ATACseq and RNAseq protocols described in “Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Reagent Kits User Guide” (see https://www.10xgenomics.com/support/single-cell-multiome-atac-plus-gene-expression/documentation/steps/library-prep/chromium-next-gem-single-cell-multiome-atac-plus-gene-expression-reagent-kits-user-guide ). TIP: The following tips pertain to the specified steps from the 10X Genomics “Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Reagent Kits User Guide”. User Guide Step 3.1 : During dynabeads cleanup, aspirate the entire supernatant including the white debris at the bottom. User Guide Steps 5.1 and 6.1 : When computing the number of PCR cycles, we round up, when within about 100 nuclei/cells of the threshold for the number of PCR cycles. User Guide Step 7.5 : When computing the number of PCR cycles, we do not round up when near the threshold for the number of PCR cycles. User Guide Step 3.1 : During dynabeads cleanup, aspirate the entire supernatant including the white debris at the bottom. User Guide Steps 5.1 and 6.1 : When computing the number of PCR cycles, we round up, when within about 100 nuclei/cells of the threshold for the number of PCR cycles. User Guide Step 7.5 : When computing the number of PCR cycles, we do not round up when near the threshold for the number of PCR cycles. QUALITY CONTROL OF ATAC LIBRARY: While we require the Agilent Bioanalyzer trace at Step 5.3 in the User Guide to contain the five expected peaks (0-nucleosome, 1-nucleosome, 2-nucleosomes, 3-nucleosomes, and multiple-nucleosomes), we are somewhat lenient in the expectation that the 1-nucleosome peak will be higher than the other peaks. If peaks are missing, we don’t proceed with the sample. If the peaks are visible but the material concentration is low, we recommend rebuilding the library from the cDNA step (Step 4.3 in the User Guide). QUALITY CONTROL OF cDNA LIBRARY: The peak of the Agilent Bioanalyzer trace at User Guide Step 6.3 should roughly be gaussian centered around 480bp. If the peak is too narrow or asymmetric, then size selection was not performed correctly and we recommend rebuilding the library from the cDNA step (Step 4.3 in the User Guide). QUALITY CONTROL OF GEX LIBRARY: The peak of the Agilent Bioanalyzer trace at User Guide Step 7.7 should roughly be gaussian centered around 480bp. If the peak is too narrow or asymmetric, then size selection was not performed correctly and we recommend rebuilding the library from the cDNA step (Step 4.3 in the User Guide). TIP: An Illumina MiSeq run can prove helpful to rebalance the libraries, if needed, and confirm the libraries are properly binding to the flow cell. MiSeq results do not have sufficient sequencing depth for downstream 10X protocol analyses.

This protocol describes H&E staining of 10X Genomics Visium slides prior to imaging and is adapted from 10X Genomics protocol documented in “Methanol Fixation, H&E Staining & Imaging for Visium Spatial Protocols” (see https://www.10xgenomics.com/support/spatial-gene-expression-fresh-frozen/documentation/steps/tissue-staining/methanol-fixation-hand-e-staining-and-imaging-for-visium-spatial-protocols ). H&E staining and imaging is essential to ensuring target tissue/region/cells are mounted within the fiducial frame of the Visium slide ( Figure 7E and 7F ). This protocol is available on protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l2owqqv1y/v1 ( Fisher et al., 2022a ). Tissue slice from OCT embedded tissue block, mounted on a 10X Visium slide Methanol, HPLC ≥ 99.9% (Milipore Sigma, cat. no. 34860) Dry ice Milli-Q water (from Milli-Q Integral Ultrapure Water System) Eosin Mix (see recipe in Reagents and Solutions ) Isopropanol (Sigma, cat. no. 563935–1L) Dako Mayer’s Hematoxylin (Lillie’s Modification) Histological Staining Reagent (Agilent, cat. no. S3309) Bluing Reagent Series S (Epredial, cat. no. 6769001) Bio-Rad T100 Thermal Cycler 10X Visium Thermocycler Adaptor (part of 10X Genomics Visium Accessory Kit; cat. no. 1000194) Prechill methanol (40mL/slide, dispensed in a 50-mL centrifuge tube) to −20°C. Place a Thermocycler Adaptor on a thermal cycler set at 37°C and equilibrate for 5 minutes. Heating the Thermocycler lid is not required. Remove slide from −80°C and place on dry ice in a sealed container. Place slide on the Thermocycler Adaptor with the active surface facing up and incubate 1 min at 37°C. CAUTION: DO NOT close the thermocycler lid. Maintain thermal cycler at 37°C. Remove slide from Thermocycler Adaptor and if necessary, wipe excess liquid from the back of the slide, without touching the tissue sections. Completely immerse the slide in the prechilled −20°C methanol. CAUTION: Secure the tube cap to prevent methanol loss. Incubate upright for 30 minutes at −20°C. Dispense the following volumes of Milli-Q water: 500mL in Beaker 1 800mL in Beaker 2 800mL in Beaker 3 800mL in Beaker 4 500mL in Beaker 1 800mL in Beaker 2 800mL in Beaker 3 800mL in Beaker 4 Dispensed volume in each beaker can be used for two slides. Prepare Eosin Mix. CAUTION: DO NOT add pure eosin to tissue sections. Remove slide from methanol and wipe excess liquid from the back of the slide, without touching the tissue sections. Place on a flat, clean, nonabsorbent work surface. Some residual droplets may remain. Add 500μL isopropanol to uniformly cover all tissue sections on the slide. Incubate 1 minute at room temperature. TIP: When incubating the slide with reagents, ensure that the slide is not in contact with any absorbent surface, like laboratory wipes, which may absorb the reagents. Discard reagent by draining and/or holding the slide at an angle with the bottom edge in contact with a laboratory wipe. Wipe excess liquid from the back of the slide, without touching the tissue sections. Place on a flat, clean, nonabsorbent work surface. Some droplets may remain. Air dry the slide for 4 minutes. Add 1mL Hematoxylin to uniformly cover all tissue sections on the slide. Incubate 5 minutes at room temperature. Discard reagent by draining and/or holding the slide at an angle with the bottom edge in contact with a laboratory wipe. Immerse the slide 5x in the water in Beaker 1. Immerse the slide 15x in the water in Beaker 2. Immerse the slide 15x in the water in Beaker 3. There is no danger of detachment unless left to soak for over 24 hours and 37°C. Wipe excess liquid from the back of the slide without touching the tissue section. Place on a flat, clean, nonabsorbent work surface. Some droplets may remain. Add 1mL Bluing Reagent Series S to uniformly cover all tissue sections. Incubate 2 minutes at room temperature. Discard reagent by draining and/or holding the slide at an angle with the bottom edge in contact with a laboratory wipe Immerse the slide 5x in the water in Beaker 3. Wipe excess liquid from the back of the slide without touching the tissue section. Place on a flat, clean, nonabsorbent work surface. Some droplets may remain. Add 1mL Eosin Mix to uniformly cover all tissue sections. Incubate 2 minutes at room temperature. Discard reagent by draining and/or holding the slide at an angle with the bottom edge in contact with a laboratory wipe. Immerse the slide 15x in the water in Beaker 4. Wipe the back of the slide with a laboratory wipe. Place on a flat, clean, nonabsorbent work surface and air dry until tissue is opaque. Incubate slide on the Thermocycler Adaptor with the thermal cycler lid open for 5 minutes at 37°C. Proceed to tissue imaging. Ensure that the entirety of the tissue slice is in the same focal plane before imaging, to reduce the risk of stitching-induced image artifacts hindering downstream analyses.

100μL Eosin Y solution (Sigma-Aldrich, cat. no. HT110216–500mL) 900μL Tris-Acetic Acid Buffer (see below) Vortex to mix Prepare fresh for each use 0.1% Nonidet P40 Substitute (Millipore-Sigma, cat. no. 74385) 10mM Trizma Hydrochloride Solution, pH 7.4 (Millipore-Sigma, cat. no. T2194) 10mM Sodium Chloride Solution, 5M (Millipore-Sigma, cat. no. 59222C) 3mM Magnesium Chloride Solution, 1M (Millipore-Sigma, cat. no. M1028) Nuclease-free water Store at 4°C and keep on ice during use The buffer will last for up to 5 days Dissolve 11g Tris base in 100mL nuclease-free water (Fisher, cat. no. BP152–500) Adjust pH to 6.0 using 100% Acetic Acid (Fisher, cat. no. A38–212) Bring volume to 200mL with nuclease-free water Filter through 0.2μm Corning 250mL Vacuum System Store at room temperature for up to 12 months This buffer is used in the Eosin Mix (see above) 1% UltraPure BSA, 50 mg/mL (Thermo Fisher Scientific, cat. no. AM2616) Protector RNase Inhibitor, 0.6U/μL (Millipore-Sigma, cat. no. 3335399001) 1X PBS (Gibco, cat. no. 14200–075) Store at 4°C and keep on ice during use The buffer will last for up to 5 days, however the RNase Inhibitor should be added the day of use

Donor selection is the first critical step for building a 3D reference map of healthy human tissue. Organ donors are uniquely positioned to facilitate the generation of a comprehensive atlas of healthy human tissue and will be the source of the ovaries, Fallopian tubes, and uteri used to generate the FRS map for HuBMAP. Criteria used to determine donor suitability must be organ-specific. For example, while including a donor with a bicuspid aortic valve would not be appropriate for investigators characterizing the heart, it would be acceptable for a group profiling the spleen. Inclusion and exclusion criteria must be explicitly defined, and all individuals involved in enrollment or recruitment of donors at the organ procurement organization must be familiar with and have access to these criteria. For this protocol, eligibility criteria for procuring FRS donor organs have been optimized to support a range of future research questions ( O’Neill, 2022 ). Donor families must be consented using Gift of Life Donor program requirements for research, and donors sex must be female. Donor must have healthy, typical, non-pregnant gross reproductive tract anatomy. Because the FRS ceases functioning at menopause (the average age of menopause in the United States US is 51 years), the University of Pennsylvania HuBMAP Tissue Mapping Center set an upper age limit of 45 years. The project set a lower age limit of 18 years, since the hypopituitary ovarian axis is still maturing in some individuals in their teens, and because some legal requirements, such as autopsies, make organ procurement difficult in individuals younger than 18 years. To ensure a structurally normal FRS, eligibility criteria exclude individuals: with uterine fibroids, endometriosis, Mullerian anomalies, and large ovarian cysts greater than 5 cm; with active hepatitis; or with a history of reproductive tract malignancy, endometriosis, or a significant uterine surgery. Because obesity can result in ovulatory dysfunction, endometrial hyperplasia, and other gynecologic pathology, individuals with body mass index over 40 are excluded. This protocol records other characteristics such as parity and current use of hormonal contraception. Trained organ procurement organization staff screen potential donors to determine if they are eligible for participation in HuBMAP research. The available legal next-of-kin of potential donors who are preliminarily suitable for organ donation via brain death are informed of donation opportunities by trained organ procurement organization staff. During review of authorization and disclosure information, a description and clarification of permitted use of donation materials (transplantation, therapy, research, and education) is addressed with the legal next-of-kin and documented appropriately. For use of donor materials in a specific research project, the study and its details should be discussed with the legal next-of-kin and included on the authorization and disclosure form. The organ procurement organization transplant coordinator then contacts the study team and the principal investigator reviews eligibility criteria. If a donor is deemed eligible, the FRS is procured as detailed below. Protected health information is removed from the donor’s medical record before the research process begins.

A comprehensive and organized approach to organ and tissue procurement is required in order to provide high quality biospecimens that can be used for scientific research. Standard operating protocols exist to sample some organs, or tissues within organs, of the female reproductive system (FRS), however a different approach must be taken when removing the FRS en bloc and when removing healthy tissue for the purposes of systematic molecular characterizaton. Finally, with the advent of molecular assays that preserve tissue architecture, the orientation and location of biospecimens within a tissue/organ must be maintained and recorded. This protocol has been developed to optimize procurement, preservation, and sampling of the organs within the female reproductive system for downstream molecular analyses. [*Copyeditor: A proper reference needs to be cited in the text below (highlighted). It also needs to be added to the Literature Cited section. Please ask the authors to provide it] Efforts to develop human tissue atlases, including but not limited to the NIH’s Human BioMolecular Atlas Program (HuBMAP), prompted the need for development of the protocols detailed in this manuscript. Organ perfusion strategies utilized for other organ systems and the advent of uterus transplantation were critical factors in developing the process for procuring the FRS en bloc from deceased donors (Protocol 1 & 2). Development of a more precise and accurate description of the anatomical regions of the ovary was recognized as a critical need and was addressed by a multidisciplinary workgroup sponsored by the Pediatric and Adolescent Gynecology arm of the NICHD (insert reference 36191605) (Protocol 3). Lastly, the importance of individualized preparation of different tissue types within the FRS for downstream molecular analysis was realized which drove the optimization and quality control checks detailed (Protocols 7 & 8). The central advantage of this approach is it provides confidence that the results obtained reflect true biologic states of healthy tissue and not pathology or artifact related to tissue/cellular degradation. The primary disadvantage is cost and the time required to execute and faithfully record all metadata concerning specimen and sample acquisition and preparation. Warm ischemic time (WIT) can compromise tissue quality and invalidate downstream molecular studies. WIT can accumulate at multiple points during acquisition of biospecimens from human tissue. Minimizing WIT requires modification of standard organ procurement protocols (if tissue is obtained from deceased donors), maintaining tissue/organs not actively being processed on ice, the use of cold blocks/molds during specimen acquisition and the use of rapid freezing protocols. Holding specimens on ice, as opposed to room temperature, has been shown to delay ischemia-induced effect for RNA analyses. Other molecular targets, such as DNA or protein, may be more robust and can tolerate additional WIT ( Johnsen et al., 2010 ). Given the importance of tissue orientation and location within a tissue/organ, it’s critical that a pathologist or similar expert review the sectioning of each biospecimen. The review can be as simple as reviewing a photograph of a histology slide and marking the region(s) of interest, so that the key molecular sectioning is appropriately oriented and positioned. The FRS contains organs with varying tissue stiffness and composition. Manual dissociation protocols that yield high quality single cell suspensions for one organ or region within an organ may not be applicable to other sites. This protocol details tissue processing workflows that were optimized in our laboratory, however quality of preparations must be examined prior to proceeding with downstream analyses in each individual laboratory. Assays that preserve tissue architecture require maintaining orientation of specimens from the point of isolation and from the organ of interest, through preparation and storage, to mounting for analysis. Standardizing the orientation of samples through these steps as well as the dyes used in the protocols are detailed above. However, we recommend verification of orientation as well as isolation of intended cell types and regions prior to proceeding with downstream analyses. All protocols may involve additional optimization. Further optimization may be needed to reduce nuclei blebbing and remove debris. For Basic Protocol 8 : Manual Tissue Dissociation for Multiome Analysis, this can include, but is not limited to, optimization of the lysis buffer incubation time, decreasing the permeabilization buffer incubation time, adjusting the Protector RNase Inhibitor concentration, and additional washes (up to 4). For Basic Protocol 9 : Tissue Dissociation for Multiome Analysis Using S2 Singulator, optimization can include, but is not limited to, titration of Protector RNase Inhibitor, addition of Digitonin to the S2 Nuclei Cartridge, premincing of tissue prior to processing in the cartridge, adjusting the disruption speed of the S2 protocol, and optimizing the number of washes. These protocols are constantly evolving and being refined as these technologies evolve and as we better understand the nuances of the tissue being investigated. We therefore recommend consulting the referenced protocol.io protocols cited in this paper, as future updates to these protocols will be documented on that site. Basic Protocols 1–6 should result in preparation of high quality biospecimens that can be used for downstream analyses. Quality of biospecimens is verified in a number of ways: Review by pathologist to ensure 1) the intended tissue is isolated and 2) there is no evidence of pathology such as malignancy or infection. For molecular assays using RNA, RNA Integrity Number (RINs) can be used to assess RNA quality and fragmentation. High-quality RNA will contain an RIN of at least 8, in general intermediate-quality RNA will contain an RIN within the range of 6–8 and samples with a RIN below 5 contain poor-quality RNA ( Schroeder et al., 2006 ). The whole process from organ procurement to initiation of downstream analyses could take up to 48 hours if done in succession. Specimen isolation and preparation must be done immediately following organ procurement, however the following protocols can be done at a later time. Time estimates for the individual protocols are detailed below: Basic Protocol 1 : Preparation and Preservation of the Female Reproductive System (ovaries, Fallopian tubes and uterus) Prior to Procurement: 15–30 minutes Basic Protocol 2 : Removal of the Female Reproductive System En Bloc : 30–45 minutes Basic Protocol 3 : Post-Surgical Dissection of Ovaries: 1 hour Basic Protocol 4 : Post-Surgical Dissection of Fallopian Tubes: 1–2 hours Basic Protocol 5 : Post-Surgical Dissection of Cervix: 30–60 minutes Basic Protocol 6 : Post-Surgical Dissection of Uterine Body: 1–2 hours Basic Protocol 7 : Tissue Slice Preparation for Visium Analysis: 15–20 minutes Basic Protocol 8 : Manual Tissue Dissociation for Multiome Analysis: 50–60 minutes Basic Protocol 9 : Tissue Dissociation for Multiome Analysis Using S2 Singulator: 50–60 minutes Support Protocol 1 : OCT Embedded Tissue Protocol: 1–2 minutes Support Protocol 2 : Tissue Fixation Protocol: 1–2 minutes Support Protocol 3 : Snap-Frozen Tissue Protocol: 1 minute Support Protocol 4 : H&E Staining for 10X Visium Imaging: 50–60 minutes

The female reproductive system (FRS) 1 – comprising the uterus, Fallopian tubes, and ovaries – undergoes some of the most dynamic changes in structure and function of any adult human organ system monthly and throughout life ( Reed & Carr, 2000 ; Strauss & Barbieri, 2019 ). These changes occur over multiple time frames: across the lifespan (i.e., pre-puberty ( Garel et al., 2001 ), during the reproductive years, and post-menopause ( Barbo, 1987 ; Pellicer et al., 1995 )); monthly during the reproductive years ( Bakos et al., 1994 ); and across the nine-month period of pregnancy ( Dickey & Hower, 1995 ; Verguts et al., 2013 ). To mediate complex functions required for human reproduction, the many diverse cell types that populate the FRS also serve as endocrine organs that regulate body metabolism and homeostasis. Cells within this organ system are also notable for rapid proliferation, angiogenesis, transformation, remodeling, and apoptosis. It is the only system in which these dynamic processes occur repeatedly in predictable monthly intervals for a large fraction of a woman’s lifespan ( Cousins et al., 2021 ; Salamonsen et al., 2021 ). Tissue organization in the FRS is paramount, since it is responsible for the coordinated cyclic changes necessary not only for reproduction but also for the proper development and function of multiple other organ systems ( Li & Wang, 2018 ; O’Kelly et al., 2022 ; Quinn & Cedars, 2018 ; Rocca et al., 2018 ; Tsiligiannis et al., 2019 ). The ovary is home for cells that determine reproductive potential (the oocyte) and cells that support oocyte development and produce the sex hormones that define the boundaries of the reproductive life span (granulosa, theca, and stromal cells). The Fallopian tubes and uterus function together to direct gametes, facilitate fertilization, promote implantation, and coordinate delivery of the fetus from a woman’s uterus during labor. Dynamic changes in the Fallopian tubes and endometrium are critical for establishing the appropriate environment for the embryo implantation and fetal development, during which time critical epigenetic markers ( Goyal et al., 2019 ; Reik & Walter, 2001 ; Waterland & Michels, 2007 ; Zhu et al., 2019 ) are established, which, in turn, may have profound and long-lasting impacts on postnatal development and adult health. The goal of the National Institutes of Health (NIH) Human BioMolecular Atlas Program (HuBMAP) is to develop an open and global platform to map the 37 trillion cells in the healthy adult human body. This ambitious program is building the framework necessary to construct the tools, resources, and cell atlases needed to determine how the relationships between cells affect health and disease. As part of this project, and building on existing single cell datasets ( Garcia-Alonso et al., 2021 ; Vento-Tormo et al., 2018 ; Wagner et al., 2020 ) our group is generating a comprehensive molecular characterization of cells of the female reproductive system, recognizing the importance of standard operating procedures for reproductive biology research ( Sheldon et al., 2011 ). We are using the following molecular assays: 10X Genomics Multiome (single cell RNAseq and single cell ATACseq) and 10X Genomics Visium spatial gene expression maps. We are collecting and managing subject/donor (deidentified) samples to generate a multi-scale molecular map, comprehensively sampling tissue from 22 specific locations across the FRS from two donors and 13 specific locations within the three organs of the FRS from an additional six donors, which will enable us to evaluate interindividual variation. Ultimately, we will use the data to generate a 3D molecular atlas of the female reproductive system. The first, critical step in analyzing the human female reproductive system is procuring organs in a manner that preserves functional characteristics. We have developed a multistep protocol that differs from existing abdominal organ harvest procedures by ensuring that the uterus and cervix are flushed ( Basic Protocol 1 ) and remain viable throughout the process of their removal ( Basic Protocol 2 ). After removal of the female reproductive system en bloc each of the organs is individually dissected ( Basic Protocols 3 , 4 , 5 , and 6 ) and preserved with OCT ( Support Protocol 1 ), PFA ( Support Protocol 2 ), formalin ( Support Protocol 2 ) or by snap freezing ( Support Protocol 3 ). The choice of preservation protocol is driven by the intended downstream use (see Figure 1 ). Here we further describe how to prepare these tissues for processing with the 10X Genomics Visium system ( Basic Protocol 7 ) using H&E staining for quality control and imaging ( Support Protocol 4 ). We additionally detail the dissociation of tissue from the different organs in preparation for processing with the 10X Genomics Multiome ATAC and RNAseq sequencing system ( Basic Protocols 8 and 9 ). These protocols detail our structured tissue sampling procedure that was designed to represent anatomical, physiological, and individual diversity, toward full exploration of the function and structure of female reproductive cells. CAUTION: All biospecimens used are by definition biohazardous or biomedical waste and therefore should be packaged, handled and disposed of in accordance with institution specific regulations. In general, solid biohazardous waste is collected in plastic autoclavable waste bags with a biohazard symbol. Double bagging and placement of bags inside an appropriately marked rigid, leak-proof container are recommended.