GoKawiilEthics statement
De-identified tissue samples were collected at Stanford University School of Medicine from elective termination of pregnancy procedures with informed consent for the research use of tissues in observance of relevant legal and institutional ethical regulations. No demographic information was collected. Consent was obtained by the medical team. The relevant tissue sample processing and analyses were performed under protocol SCRO-796, approved by the Stem Cell Research Oversight Panel (SCRO) at Stanford.
Sample collection and nuclei isolation
Tissue samples were delivered on ice and immediately stored in liquid nitrogen prior to processing. A multi-tissue compatible nuclei isolation protocol was developed to efficiently isolate stable nuclei for further library preparation. In brief, for a given sample, 100–200 mg of tissue was added directly into 1 ml of Nuclei Extraction Buffer (250 mM Sucrose, 25 mM KCl, 5 mM MgCl 2 , 20 mM HEPES-KOH, 65 mM β-glycerol, 0.5% IGEPAL CA-630, 1× protease inhibitor, 1 mM DTT, 0.2 mM Spermine, 0.5 mM Spermidine, 60 U ml−1 RNasin Plus, 2–5% normal goat serum) in a chilled 2 ml dounce homogenizer (Kimble 885300-0002) on ice. The sample was incubated for 10 min on ice. The sample was dounced 20 times each with pestle A then with pestle B. Sample was transferred to a DNA low binding tube. Three hundred µl additional Nuclei Extraction Buffer was used to rinse any remaining nuclei from dounce homogenizer. Sample was incubated with vertical rotation for 5 min at 4 °C. Sample was filtered using a 70-µm Flowmi strainer. Volume was adjusted with additional Nuclei Extraction Buffer to 1.2 ml total volume. Thirty-seven per cent formaldehyde was added to the sample for a 0.2% final formaldehyde concentration and incubated for 4 min at room temperature with vertical rotation. Fixation was quenched with 125 mM glycine for 8 min at room temperature with vertical rotation. Nuclei Extraction Buffer was added to the sample for a final volume of 1.4 ml. An iodixanol gradient was prepared to enrich nuclei from homogenate. In brief, 50% iodixanol solution was prepared from 60% iodixanol with the addition of 1 mM DTT, 60 U ml−1 RNasin Plus, and 2–5% normal goat serum. The sample was mixed with an appropriate amount of iodixanol for a final 22% iodixanol concentration. 44% iodixanol solution was layered below the sample. Then, a 22% iodixanol solution was gently added between the sample and the 44% iodixanol solution layer. The sample was centrifuged at 3,500g for 30 min at 4 °C with brakes off. The nuclei layer was separated with gentle pipetting for further processing.
SHARE-seq library preparation
The full protocol is described in Supplementary Note 1, adapted from published SHARE-seq protocols21,91. In total, we processed 76 tissue samples derived from 23 individuals, across 12 tissue processing and SHARE-seq library preparation batches, where each batch corresponded to all samples of a given organ.
Library sequencing
All DNA libraries were sequenced on a NovaSeq 6000 using 300-cycle S4 v1.5 reagent kits with XP workflow. Paired-end sequencing was run with a 96-99-8-96 configuration (Read1-Index1-Index2-Read2). We quantified DNA libraries using Qubit and Tapestation, then prepared library pools at 1.5 nM concentration for a final loading concentration of 300 pM. Sequencing was performed at the Stanford Genome Technology Center.
VISTA embryo histology
We received X-Gal-stained and fixed whole mouse embryos in PBS from L. Pennachio26,27 and transferred them to 70% ethanol for storage. Paraffin embedding was performed by Histo-Tec Laboratory using a xylene-free dehydration protocol as xylene could dissolve the X-Gal stain. In brief, the embryos were sequentially dehydrated with 80%, 95%, 100%, 100% and 100% ethanol for 20 min each, followed by washes with 50:50, 80:20, 90:10 and 100:0 paraffin:alcohol mix for 20 min each to remove the ethanol. Subsequent embedding and H&E staining was performed with standard protocols on 5-μm sections.
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