A Multi-omic Atlas of Human Embryonic Skeletal Development -Multiome GEX+ATAC
A Multi-omic Atlas of Human Embryonic Skeletal Development -Multiome GEX+ATAC
Developing human limb and cranium tissue samples were obtained from elective terminations under REC 96/085 (East of England - Cambridge Central Research Ethics Committee). Briefly, samples were kept suspended in PBS and at -4°C on ice during dissection. Shoulder, hip and knee joints were dissected en-bloc from the limbs. For the shoulder joint a proximal incision was made at the distal third of the clavicle, and a distal incision was created at the neck of the humerus. For embryonic shoulder samples where distinctive bone features had not formed, approximations were made to capture the entirety of the glenohumeral and acromioclavicular joints. For the cranium samples (<8 PCW), two regions were dissected for each of the calvaria and skull base, separated at the posterior border of the frontal bone in both cases. For older cranial samples (>8 PCW), tissues were dissected to separate the frontal, parietal, sphenoid, ethmoid, occipital and temporal bones where feasible. Samples were initially embedded in optimal cutting temperature medium (OCT) and frozen at -80°C on an isopentane-dry ice slurry. Cryosections were then cut at a thickness of 10 μm using a Leica CM1950 cryostat and placed onto SuperFrost Plus slides (VWR) for ISS or Visium CytAssist, or used directly for single-nuclei processing. For whole-mount immunostaining, samples used in this study were obtained from terminations of pregnancy with written and informed consent from all sample donors. Samples were provided by INSERM's HuDeCA Biobank and utilised in compliance with French regulations. Authorization to use these tissues was granted by the French agency for biomedical research (Agence de la Biomédecine, Saint-Denis La Plaine, France; N° PFS19-012) and the INSERM Ethics Committee (IRB00003888). Single-nuclei were isolated from fresh frozen samples through cryosectioning followed by mechanical dissociation as described in previous work 135. Briefly, 10 μm sections were homogenised in homogenization buffer (250 mM sucrose, 25 mM KCl, 5 mM MgCl2, 10 mM Tris-HCl, 1 mM dithiothreitol (DTT), 1× protease inhibitor, 0.4 U μl−1 RNaseIn, 0.2 U μl−1 SUPERaseIn and 0.1% Triton X-100 in nuclease-free water) using a glass Dounce tissue grinder set (Merck). Samples were dissociated with 10-20 strokes of a loose pestle 'A' followed by 10 strokes of a tight pestle 'B' when tissue fragments remained. The resulting mixture was passed through a 50 μm cell strainer, followed by centrifugation (500 g, 5 mins) the pellet was then resuspended in 300 μl of storage buffer (1× PBS, 4% BSA and 0.2 U μl−1 Protector RNaseIn) and passed through a 20 μm cell strainer.  Nuclei were visualised and assessed for viability under microscopy following staining with trypan blue solution and were further processed for 10x Genomics Chromium Single Cell Multiome ATAC + Gene Expression according to the manufacturer's protocol. Nuclei suspensions were loaded with a targeted nuclei recovery of 16,000 droplets per reaction. For some of the nuclei samples, mixtures of samples from different sample donors were pooled within one reaction and later demultiplexed by genotype. Quality control of cDNA and final libraries was done using Bioanalyzer High Sensitivity DNA Analysis (Agilent). 