GSM5513652: Uterus_EZKO_rep4; Mus musculus; RNA-Seq
One Ezh2cKO and one WT female randomly selected from four different litters. Females were ovariectomized at postnatal day (PND) 71 ± 4. Uteri were collected on PND 112±11. Uteri were embedded and frozen in Tissue-Tek optimal cutting temperature (O.C.T.) compound (Sakura Finetek USA, #25608-930). Samples were cut using a Thermo Scientific HM525 NX cryostat, and 10 µm thick sections were placed on capture areas of Visium spatial gene expression slides (10X Genomics). Immediately after sectioning, tissues were fixed and stained according to manufacturer's recommendations. In brief, tissue sections were fixed in prechilled methanol for 30 min at -20°C, stained with Mayer hematoxylin for 7 min followed by washing in 3 changes of nuclease-free water and a 2 min incubation in bluing buffer (Dako, Agilent, Santa Clara, CA, United States). Subsequently, sections were counterstained with eosin for 1 min, then briefly washed and dried for 5 min on a slide warmer at 37ºC. Images of individual capture areas with stained sections were acquired using a Zeiss Axiovert 200M motorized microscope equipped with a 5x Plan Apochromat objective and a Leica Buffalo Grove, IL United States DFC290 camera. Image tiles were stitched with Metamorph 7.8.12 software. Pixel size of final composite images was 1.275 µm. The cDNA library for the Illumina platform was prepared by following the 10X Genomics spatial gene expression library construction protocol. Briefly, double stranded cDNA was fragmented, end repaired, and A-tail added in a single step. The A-tailed fragments were size selected using double-sided AxyPrep Mag magnetic beads with a bead to reaction solution ratio of 0.6X and 0.8X for the for the first and second steps, respectively. Next, the adaptor was ligated and purified with 0.8X AxyPrep Mag magnetic beads. The final Illumina libraries were amplified by PCR, which added unique dual indexes to each sample. The PCR-amplified libraries were purified and size selected by using two reactions with double-sided AxyPrep Mag magnetic beads at the bead to reaction solution ratios described above. The individual library was analyzed by Fragment Analyzer (Agilent, Santa Clara, CA, United States) to determine the quality and average fragment size and quantified by Qubit (Invitrogen, Waltham, MA, United States) DNA assay. The libraries were pooled in an equimolar ratio with a total concentration of 5 nM and sequenced on the Illumina Novaseq 6000. Spatial transcriptomics