GSM6398390: Cit+ E7.5 Decidua, scRNAseq; Mus musculus; RNA-Seq
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 washed and resuspended in RPMI + 10% FBS and stained with DAPI. Live Citrine+ were sorted using a FACSAria II cell sorter according to the presence or absence of Citrine signal using a C57BL6 mouse as negative control. Citrine+ cells were washed in RPMI + 10% FBS and resuspended in PBS. Cell number and viability were determined by a Propidium Iodide based fluorescence assay using Moxi GO II System, Orflo Prod#MXG102 (ORFLO Technologies, LLC). Single-cell partitioning and single-cell RNA-Seq libraries were generated using the 10X Chromium Controller and the Chromium Single-Cell 3′ Reagent v3 Kit (10X Genomics, Pleasanton, CA) as per the manufacturer's protocol. Briefly, live cells in single cell suspension with a >90% viability were mixed with RT reagents and loaded onto chromium Chip B 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 Gel Beads-in-Emulsion (GEMs) 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. 3' Gene expression libraries were prepared by following the Chromium NextGEM Single-Cell 3' Reagent Kits v3 user guide. Library quantity and quality control were performed using Qubit Fluorometer (London, England) and TapeStation (Agilent Technologies, Santa Clara CA). Libraries were sequenced on Illumina NovaSeq 6000 system (Illumina, San Diego, CA), with a configuration of 28/90 bp as recommended by 10X Genomics.