Tapestation high sensitivity d5000 kit

To profile single cells/nuclei from three different areas of the human myometrium (anterior, posterior, and fundus), scRNA-seq analysis was performed using the 10X Chromium system (10X Genomics #1000204). Approximately 17,000 cells or nuclei were loaded onto a 10X G Chip to obtain Gel Bead-in-emulsions (GEMs) each containing an individual cell. GEMs were used to generate barcoded cDNA libraries following the manufacturer’s protocol (Single Cell 3’ Reagent Kit v3.1, 10X Genomics, #PN-1000268) and quantified using the TapeStation High Sensitivity D5000 kit (Agilent, #5067-5593). Subsequently, gene expression libraries were constructed using 1–100 ng of each amplified cDNA library and quantified using the TapeStation High Sensitivity D1000 kit (Agilent, #5067-5584) to determine the average fragment size and library concentration. Libraries were normalized, diluted, and sequenced on the Illumina NovaSeq 6000 system (Illumina) according to the manufacturer’s instructions.

Isolated DNA integrity was determined using a multiplex GAPDH PCR approach outlined by van Beers et al¹⁴ (link) using 2 ng of DNA input, 12.5 μL of AmpliTaq Gold DNA Polymerase (Thermo Fisher Scientific), and 10 pmol of each primer (Integrated DNA Technologies) in a total reaction volume of 25 μL. PCR products were analyzed on a 2.6% agarose gel with GelRed Nucleic Acid Gel Stain (Biotium). During library preparation, samples with low integrity (≤2 amplification lines) were amplified with 2 additional PCR cycles compared with the vendor’s recommendation. Formalin fixation–induced DNA damages were repaired using the NEBNext FFPE DNA Repair Kit (New England Biolabs). Sequencing libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit for Illumina with NEBNext Unique Dual Index UMI Adaptors (New England Biolabs). After quantification and quality control with the Qubit HS dsDNA Kit (Qiagen) and TapeStation High Sensitivity D5000 Kit (Agilent), libraries were pooled equimolarly and loaded on a NextSeq2000 (Illumina) P2 or P3 flow-cell for 50 cycle, single-read sequencing with a target yield of 10 M bases per library. Sequencing metrics and genome-wide copy number burden results are summarized in Supplementary Table S1.