GSM6704955: F1_Maternal; Mus musculus; RNA-Seq
Placenta Blood: N=4 non-sister female mice at GD10.5 were processed per group (WT and Het). After terminal anesthesia by pentobarbital, maximum peripheral whole blood (~2 mL) was collected via cardiac puncture. Biological replicates from each group were then pooled together and an equal volume of 1X DPBS without magnesium or calcium was added to the whole blood. The diluted whole blood was layered onto Ficoll-Paque PREMIUM 1.084 (2 parts blood to 3 parts Ficoll in volume). The gradient was spun at 400 x g for 30 min with the brakes off at room temperature (RT). Plasma was removed and the layer containing peripheral blood mononuclear cell (PBMC) was collected and transferred to a new tube. The remaining Ficoll layer was removed, and the RBC/granulocyte pellet was suspended in 1X RBC Lysis Buffer in a 1:10 ratio. The solution was gently inverted a few times, incubated at RT for 15 minutes, then spun at 300 x g for 5 minutes with brakes on low at RT. The supernatant with lysed RBC fragments was removed. The pellet was washed with a volume of DPBS equal to the amount of RBC Lysis Buffer used and spun at 350 x g with brakes on low for 5 minutes at RT. Wash was repeated, the supernatant was removed, and the resulting pellet of granulocytes was resuspended in 1 mL DPBS with 2% FBS and set on ice. Separately, the volume of collected PBMC was estimated and 4X that volume of DPBS was added to the PBMC fraction. The sample was gently triturated with a serological pipet, then spun at 400 x g for 10 minutes with brakes on low at RT. RBC lysis buffer was added to the pellet as above and the resulting pellet was washed twice with DPBS. Final PBMC pellet was resuspended in 1 mL DPBS with 2% FBS, combined with granulocyte suspension, and visually inspected with a hemocytometer under a microscope for cell counting. Placenta: After dissection of the maternal decidua, N=3 decidua from non-sibling placenta at GD10.5 were processed per group (WT and Het decidua from WT and F1 placenta). Single nuclei suspension was prepared from frozen tissue with slight modifications to the method described by Marsh and Blelloch 2020 (Marsh and Blelloch 2020). In brief, 1 mL prechilled Nuclei EZ lysis buffer was added to each flash-frozen decidua sample, which was then minced finely with dissection scissors. A wide bore pipet was used to transfer pooled minced tissue into a glass dounce homogenizer. Tissue was lysed with 20 strokes of the loose pestle followed by 20 strokes of the tight pestle. The homogenate was transferred to a new tube and an additional 3 mL of prechilled Nuclei EZ Lysis Buffer was added. The sample was incubated on ice for 5 min and was resuspended a few times throughout. The lysate was filtered through a 70 µM filter and spun at 500 x g for 5 min at 4°C in a centrifuge with a swinging bucket rotor. The supernatant was removed, and the pellet was resuspended with 2 mL Nuclei EZ Lysis Buffer and incubated for an additional 5 min on ice. The sample was spun at 500 x g for 5 min at 4°C and the supernatant was removed. 1 mL Nuclei Wash Buffer (DPBS without magnesium and calcium, 2% BSA, and 0.2U Protector RNase inhibitor was layered onto the pellet and the sample was incubated on ice for 5 min without mixing or resuspending in order to promote buffer exchange. An additional 2 mL Nuclei Wash Buffer was used to resuspend, and the sample was spun at 500 x g for 5 min at 4°C. The lysate was removed, and this wash step was repeated. Finally, the pellet was resuspended in 1.5 mL Nuclei Wash Buffer, filtered through a 70 µM filter, and visually inspected with a hemocytometer under a microscope for cell counting. Libraries were constructed according to manufacterer protocols by the Weill Cornell Epigenomics Core using the Chromium Single Cell 3' Reagent V3 kit.