c-Myc supports polyploidy and prevents senescence in the murine placenta [scRNA-Seq]

Placenta is essential for reproductive success and placental abnormalities are the leading cause of low birth weight and preterm birth. The placenta includes polyploid cells that are crucial for its function. Polyploidy occurs broadly in nature but the regulators that enable polyploidy to arise in a spatiotemporally regulated manner in the placenta are unknown. We used scRNA seq to understand molecular regulators of polyploidy. 14.5 dpc placenta was isolated in D-PBS (Thermo Fisher Scientific (#14287072) and adult brain dissociation kit (#130-107-677) was used to make single cell suspension as per manufacturer instructions. Each 14.5 dpc placenta was cut into four small pieces using scalpel blades. Placental pieces were transferred in gentleMACS C tube (Miltenyi Biotec- #130-093-237) and 1950 u­l of Enzyme mix 1 (Buffer Z 1900 ul + Enzyme P 50 ul) was added. Further 30 ul of Enzyme mix 2 (Buffer Y 20 ul + Enzyme A 10 ul to the same tube was added. 37C_ABDK_01 program of gentleMACS was used and once program is completed, cells were centrifuged at 300Xg for 5 minutes. Pellet was resuspended in cold 10 ml D-PBS and filtered it through 70 u­m MACS SmartStrainer (Miltenyi Biotec- #130-098-462) using 50 mL falcon tube. C tube was washed again with 10 ml cold D-PBS and filter with the same SmartStrainer. MACS SmartStrainer was discarded and cell suspension was centrifuged at 300Xg for 10 minutes at 4°C. Supernatant was removed and pellet was resuspended in cold 1550 ul D-PBS and transferred it to 15 mL falcon tube. 450 ul of debris removal solution was added and mixed well. Gently 2 mL of cold D-PBS was overlayed, and tube was centrifuged at 4°C and 3000Xg for 10 minutes. Three phases were formed after centrifugation. Top two layers were discarded, and tube was filled with 10 mL cold D-PBS. Tube was gently mixed and centrifuged at 4°C and 1000Xg for 10 minutes. Pellet was resuspended in 0.5 mL of 1X red blood cell removal solution and incubated for 10 minutes at 4°C. 5 mL of cold -DPBS was added and centrifuged at 4°C and 300Xg for 10 minutes. Pellet was resuspended in 0.5 ml of cold PBS and acridine orange (AO)/ propidium iodide (PI) method was used to cell viability. Samples with viability above 87% were used for further sequencing. Chromium Next GEM Single Cell 3' Reagent Kits v3.1 was used for making cDNA library using 10X Genomic reagents (Next GEM Chip G Single Cell Kit #1000120, Next GEM Single Cell 3' Gel Bead Kit v3.1 #1000122, Single Cell 3' Gel Beads v3.1#2000164, Next GEM Single Cell 3' GEM Kit v3.1 #1000123, Next GEM Single Cell 3' Library Kit v3.1 #1000157, i7 Multiplex Kit #120262, DynaBead - MyOne Silane Beads #2000048 and SPRIselect Beads #17201600). Sequencing was performed using Illumina Nova-Seq-SP.胎盘对生殖成功至关重要，而胎盘异常是低出生体重与早产的首要诱因。胎盘含有对其功能至关重要的多倍体细胞。多倍化在自然界广泛存在，但胎盘内以时空调控方式引发多倍化的分子调控因子仍未明确。本研究采用单细胞RNA测序（scRNA-seq）探究多倍化的调控机制。取14.5天胎龄（days post coitum, dpc）的胎盘，置于杜氏磷酸缓冲盐溶液（D-PBS，赛默飞世尔科技，货号#14287072）中分离；按照制造商说明书，使用成年脑组织解离试剂盒（#130-107-677）制备单细胞悬液。将每块14.5 dpc的胎盘用手术刀片切成4小块，转移至gentleMACS C管（美天旎生物，货号#130-093-237），加入1950 μL酶混合液1（Z缓冲液1900 μL + P酶50 μL），随后再向同一管中加入30 μL酶混合液2（Y缓冲液20 μL + A酶10 μL）。启动gentleMACS的37C_ABDK_01程序，程序结束后将细胞以300×g离心5分钟。将沉淀重悬于预冷的10 mL D-PBS中，使用50 mL离心管通过70 μm MACS SmartStrainer过滤器（美天旎生物，货号#130-098-462）过滤细胞悬液；再次用10 mL预冷D-PBS冲洗C管，并用同一过滤器过滤洗液。弃去过滤器，将细胞悬液以4℃、300×g离心10分钟，弃去上清，将沉淀重悬于1550 μL预冷D-PBS中并转移至15 mL离心管。加入450 μL杂质去除液并充分混匀，小心铺上2 mL预冷D-PBS，随后以4℃、3000×g离心10分钟。离心后形成三层液体，弃去最上层两层，向管中加入10 mL预冷D-PBS，轻柔混匀后以4℃、1000×g离心10分钟。将沉淀重悬于0.5 mL 1×红细胞去除液中，4℃孵育10分钟；加入5 mL预冷D-PBS，以4℃、300×g离心10分钟。将沉淀重悬于0.5 mL预冷PBS中，采用吖啶橙（AO）/碘化丙啶（PI）染色法检测细胞活性，选取活性高于87%的样本用于后续测序。采用10×基因组学（10X Genomics）配套试剂，通过Chromium Next GEM单细胞3'端转录组试剂盒v3.1构建cDNA文库，所用试剂包括：Next GEM Chip G单细胞试剂盒（#1000120）、Next GEM单细胞3'端凝胶磁珠试剂盒v3.1（#1000122）、单细胞3'端凝胶磁珠v3.1（#2000164）、Next GEM单细胞3'端GEM试剂盒v3.1（#1000123）、Next GEM单细胞3'端文库试剂盒v3.1（#1000157）、i7多重试剂盒（#120262）、DynaBeads MyOne硅烷磁珠（#2000048）以及SPRIselect磁珠（#17201600）。测序实验采用Illumina NovaSeq-SP测序平台完成。