title: GSM8371716: S38 at embryonic day 12.5, snRNA-seq; Mus musculus; RNA-Seq
library_construction_protocol: Nuclei were isolated following a protocol detailed by Rousselle et al. (3) for human kidney biopsies, which we modified to suit mouse placental and decidual samples. Each sample was minced into roughly 1 mm pieces and returned to their original centrifuge tubes. A 7 mm micro stir-bar was placed in each tube, along with 1 ml lysis buffer that was composed of 1× PBS pH, 7.4 10 mM Tris–HCL pH 7.5,0.0125% Triton X-100, 1 mM dithiothreitol, and 0.2 U/mL RNAse inhibitor (Promega, cat. no. PRN2611), and 1.5 l DNAse I, RNase free (1U/l, Thermo Fisher Scientific, cat. no. AM2222). The samples were then placed on ice on a magnetic stir plate and stirred at ~350 RPM for 10 min. The contents were allowed to settle, and the supernatant solution removed by gentle suction with a P1000 pipette. The homogenate was passed through a 100 m filter into a 15 ml collection tube and the filter washed with 6 ml nuclear wash and resuspension buffer (1x PBS, pH 7.4, 2 % bovine serum albumin [Sigma-Aldrich, cat. no. A9418-50G], and 0.2U/ml RNAse inhibitor). The process was repeated twice on the original tissue samples, and the three preparations combined, re-centrifuged, resuspended, and filtered through a 100 m filter. The pelleted nuclei were resuspended in 0.2 ml of ice-cold buffer and pipetted 50 times to minimize nuclear aggregation. Next, the isolated nuclei samples were transferred to a 1.5 mL microcentrifuge tube and were purified with Optiprep as detailed in Martelotto's protocol (4), which is recommended by Parse Biosciences. Isolated nuclei were fixed with Evercode™ Nuclei Fixation v2.  Nuclei barcoding, cDNA synthesis, and library preparation were performed with the Parse Evercode™ WT v2 kit. Final library sequencing The size and purity of the final library was determined with the Fragment Analyzer (Agilent Technologies, Inc.), quantified with the Qubit dsDNA HS Assay kit (Invitrogen), and diluted according to Illumina's standard sequencing protocol. Libraries were pooled and run on an Illumina NovaSeq 6000 sequencer by using a paired end 100 bp read format on a S4 flow cell to generate ~100 million paired reads per sample. snRNA-seq