title: GSM8636771: PD21 CTRL, pooled, scRNAseq; Mus musculus; RNA-Seq
library_name: GSM8636771
library_construction_protocol: Uterine horns were longitudinally incised and segmented into 2-3 mm pieces following dissection. These tissue fragments were then enzymatically digested in 15-ml conical tubes containing 1% trypsin in Ca2+- and Mg2+-free Hank's Balanced Salt Solution (CM-HBSS) at room temperature for one hour with agitation. To terminate the digestion, an equal volume of fetal bovine serum (FBS) was added, and the tubes were vigorously shaken to dislodge uterine cells. The supernatant, which contained the dissociated cells, was carefully pipetted out and passed through 70 µm nylon filters to remove debris. The filtered cells were washed twice, collected by low-speed centrifugation, and subsequently resuspended in 0.04% bovine serum albumin (BSA) in 1x PBS at a concentration of 1000 cells/µl, with cell viability exceeding 85%. The cell suspension was then sent to the McDonnell Genome Institute (MGI) at Washington University School of Medicine for single-cell RNA sequencing. cDNA was prepared after the GEM generation and barcoding, followed by the GEM-RT reaction and bead cleanup steps.  Purified cDNA was amplified for 11-16 cycles before being cleaned up using SPRIselect beads.  Samples were then run on a Bioanalyzer to determine the cDNA concentration.  Gene Expression libraries were prepared as recommended by the 10x Genomics Chromium Chromium Single Cell 5' Reagent Kits User Guide (v2 Chemistry Dual Index user guide, 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 5' Kit v2, 16 rxns (PN-1000263), Chromium Next GEM Chip K Single Cell Kit, 48 rxns (PN-1000286), Dual Index Kit TT Set A, 96 rxns (PN-1000215) were used.  The concentration of each library was accurately 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 151x10x10x151 sequencing recipe according to the manufacturer protocol.  A median sequencing depth of 50,000 reads/cell was targeted for each Gene Expression library.
identifiers.namespace: GEO
identifiers.id: GSM8636771_r1