GSM6398393: CD45+ E7.5 Decidua, scRNAseq, Replicate 2; Mus musculus; RNA-Seq
C57BL6 (Hashtag A0306) and Il33cit/cit (IL-33-deficient) (Hashtag A0309) dams were mated with C57BL6 studs. At E7.5, the decidua and myometrium were mechanically isolated and minced with scissors. Tissues were digested in RPMI-1640 + 1 mg/mL Collagenase A + 0.1 mg/mL DNase I in a shaker incubator at 250 rpm at 37ºC for 2x12 min (decidua) or 2x15 min (myometrium). After each cycle, tissues were mechanically dissociated by passage through 18G or 20G needle. Single cells suspension was filtered through 100 um strainer and washed with cold RPMI + 3% FBS. Red blood cell lysis was performed. Cells were enriched using anti-Podoplanin-Biotin antibody and Dead Cell Removal (Annexin V) kit, then stained separately with TotalSeq-A cell hashing  (BioLegend) antibodies. Cells were washed and re-suspended in RPMI + 10% FBS and stained with a DAPI. Live CD45.2+ cells were isolated by fluorescence-activated cell sorting (FACS) using a FACSAria II cell sorter. Sorted cells from decidua or myometrium were then pooled and resuspended for scRNA-seq analysis. Cell number and viability were determined by a Propidium Iodide based fluorescence assay using Moxi GO II System, Orflo Prod#MXG102 (ORFLO Technologies, LLC). Cells hash tagged with TotalSeq-A antibodies were pooled and the GEMs (Gel Beads-in-Emulsion) were generated using the Chromium Single Cell 3ʹ Reagent Kits Next Gen v3.1 (10X Genomics, Pleasanton, CA) and by following the manufacturer's protocol. Briefly, hash-tagged live cells in single cell suspension with a >90% viability were mixed with RT reagents and loaded onto Chromium Chip G along with Gel beads and Partitioning oil in the recommended order and then the chip was processed through 10X Chromium Controller for the generation of GEMs, followed by RT, Cleanup, and cDNA amplification. After the cDNA amplification, size selection was used to separate the cDNA molecules for 3' Gexp (Gene Expression) library and HTO (Hash Tag Oligonuclotide) library construction. 3' Gene expression libraries were prepared by following the Chromium NextGEM Single-Cell 3' Reagent Kits v3.1 User Guide. HTO libraries were prepared using the TotalSeq-A antibodies and Cell Hashing with 10X Single Cell 3' Reagent kit v3.1 (Dual Index) Protocol (BioLegend, San Diego, CA). Library quantity and quality control were performed using Qubit Fluorometer (London, England) and TapeStation (Agilent Technologies, Santa Clara CA). HTO and 3'Gexp libraries were mixed at the ratio of 1:5 and sequenced on Illumina NovaSeq 6000 system (Illumina, San Diego, CA), with a configuration of 28/90 bp as recommended by 10X Genomics.  The Chromium Single Cell Software is used to analyze and visualize single cell 3' RNASeq data produced by the 10X Chromium Platform.  The 10X chromium software package includes Cell Ranger Pipelines and Loupe Cell Browser.  Cell Ranger pipelines use raw 10X single cell sequencing data from an Illumina sequencer and perform demultiplexing, unique barcode processing, and single cell 3' gene counting. Loupe Cell Browser uses data output from the Cell Ranger pipelines for fast analysis of significant genes, cell types, and substructure using single cell data. scRNA-seq with HTOs