GSM8322904: WTZ6DPI,4,S8; Mus musculus; RNA-Seq
Two placentas from the same genotype and condition were pooled and processed for isolation of nuclei. Nuclear extraction was adapted from a previously described protocol for jejunal segments (Chang et al., 2024). Briefly, tissues were thawed and minced in lysis buffer (25 mM citric acid, 250 mM sucrose, 0.1% NP-40, and 1X protease inhibitor (Roche)). Nuclei were released from cells using a Dounce homogenizer (Wheaton) then washed 3 times with buffer (25 mM citric acid, 0.25 M sucrose, 1X protease inhibitor (Roche)), and then filtered successively through 100 μm, 70 μm, and 40 μm diameter strainers (pluriSelect) to obtain single nuclei in resuspension buffer (25 mM KCl, 3 mM MgCl2, 50 mM Tris, 1 mM DTT, 0.4 U/μL RNase inhibitor (Sigma) and 0.4 U/μL Superase inhibitor (ThermoFisher)). Approximately 10,000 nuclei per sample were subjected to gel bead-in-emulsion (GEM) generation, reverse transcription, and construction of libraries for sequencing according to the protocol provided in the 3' gene expression v3.1 kit manual (10X Genomics). Briefly, cDNA was prepared after GEM generation and barcoding, followed by the GEM-RT reaction and bead cleanup steps. Purified cDNA was amplified for 11-13 cycles before clean-up using SPRI select beads (Beckman Coulter, B23318). Samples were then run on a Bioanalyzer to determine cDNA concentrations. Gene expression libraries (GEX libraries) were prepared as recommended by the 10x Genomics Chromium Single Cell 3' Reagent Kits User Guide (v3.1 Chemistry Dual Index) with appropriate modifications to the PCR cycles based on the calculated cDNA concentration. For sample preparation on the 10x Genomics platform, the Chromium Next GEM Single Cell 3' Kit v3.1, 16 rxns (PN-1000268), Chromium Next GEM Chip G Single Cell Kit, 48 rxns (PN-1000120), and Dual Index Kit TT Set A, 96 rxns (PN-1000215) were used.  The concentration of each library was determined through qPCR utilizing the KAPA library Quantification Kit according to the manufacturer's protocol (KAPA Biosystems/Roche) to produce cluster counts appropriate for the Illumina NovaSeq6000 instrument. Normalized libraries were sequenced on a NovaSeq6000 S4 Flow Cell using the XP workflow and a 50x10x16x150 sequencing recipe according to manufacturer protocol. A median sequencing depth of 50,000 reads/cell was targeted for each Gene Expression Library.