title: GSM6883169: Dnmt3b WT_E12.5 placenta; Mus musculus; RNA-Seq
library_construction_protocol: Natural timed matings between homozygous Dnmt3b floxed females and heterozygous Dnmt3b del, Sox2-Cre +ve males were used to generate cKO and WT embryos. E12.5 placentas were collected and a tail clip was used for genotyping. Single nuclei were isolated as described in the 'Frankenstein' protocol, available through 10X Genomics (https://www.10xgenomics.com/resources/customer-developed-protocols) and published adaptations for mouse placenta. Following collection and genotyping, Dnmt3b KO and WT E12.5 placentas were processed in parallel immediately for single nuclei isolation and 10X Genomics RNA-seq preparation. Placentas were first chopped with a razor blade in 1mL nuclei EZ lysis buffer (Sigma) for 2 mins, followed by homogenisation in a Dounce homogenizer. Nuclei were filtered through a 30µm filter, and then pelleted by centrifugation at 500g for 5 mins at 4°C. Nuclei were resuspended in fresh nuclei EZ lysis buffer, pelleted and then resuspended in nuclei wash and resuspension buffer (1% BSA, 0.2U/µL RNase Inhibitor, PBS). Nuclei were gently resuspended, pelleted and resuspended in fresh nuclei wash and resuspension buffer. Nuclei were passed again through a 30µm filter, before visual inspection of nuclei with a haemocytometer. Nuclei were then stained with DAPI (adding 1 µL of 1mg/mL DAPI) before a total of 10,000 nuclei per sample were flow sorted using the BD FACSAria Fusion sorter with 70µm nozzle into RT reaction mix (10X Genomics). Samples were immediately processed to cDNA using the Chromium Controller (10X Genomics), followed by 3' single-nuclei RNA-seq library preparation (10X Genomics).  Libraries were multiplexed for sequencing on an Illumina NextSeq500. Natural timed matings between heterozygous females and males were used for the Dnmt3b KO strain to generate KO and WT embryos or between homozygous Dnmt3b floxed females and heterozygous Dnmt3b del, Sox2-Cre +ve males were used to generate cKO and WT embryos. E12.5 placentas were collected and a tail clip was used for genotyping. Single nuclei were isolated as described in the 'Frankenstein' protocol, available through 10X Genomics (https://www.10xgenomics.com/resources/customer-developed-protocols) and published adaptations for mouse placenta. Following collection and genotyping, Dnmt3b KO and WT E12.5 placentas were processed in parallel immediately for single nuclei isolation and 10X Genomics RNA-seq preparation. Placentas were first chopped with a razor blade in 1mL nuclei EZ lysis buffer (Sigma) for 2 mins, followed by homogenisation in a Dounce homogenizer. Nuclei were filtered through a 30µm filter, and then pelleted by centrifugation at 500g for 5 mins at 4°C. Nuclei were resuspended in fresh nuclei EZ lysis buffer, pelleted and then resuspended in nuclei wash and resuspension buffer (1% BSA, 0.2U/µL RNase Inhibitor, PBS). Nuclei were gently resuspended, pelleted and resuspended in fresh nuclei wash and resuspension buffer. Nuclei were passed again through a 30µm filter, before visual inspection of nuclei with a haemocytometer. Nuclei were then stained with DAPI (adding 1 µL of 1mg/mL DAPI) before a total of 10,000 nuclei per sample were flow sorted using the BD FACSAria Fusion sorter with 70µm nozzle into RT reaction mix (10X Genomics). Samples were immediately processed to cDNA using the Chromium Controller (10X Genomics), followed by 3' single-nuclei RNA-seq library preparation (10X Genomics).  Libraries were multiplexed for sequencing on an Illumina NextSeq500. single-nuclei RNA-seq