Single cell 3 reagent kit v3

The single-cell suspensions collected from the above step were used for scRNA-seq library construction following the instruction of the Single Cell 3′ Reagent Kit v3.1 (10× Genomics, PN-1000121, Pleasanton, CA, USA). The scRNA-seq libraries were constructed using the Chromium Next GEM Single Cell 3′ Kit v3.1 from 10× Genomics, according to the manufacturer's instructions. Briefly, single cells were diluted to a final concentration of 1,000 cells/μL. Then, approximately 10,000 cells were captured in droplets to generate nanoliter-scale Gel beads in EMulsion (GEMs). Then, GEMs were reverse transcribed before cell barcoding, the emulsions were broken, and the cDNA was separated and further purified before PCR amplification. Next, the obtained amplified cDNA was used for library construction. To build-up the RNA-seq library, the amplified cDNA was fragmented and end-repaired, followed by double-sided size selection and PCR amplification with sample index primers. The libraries were further purified and profiled for quality evaluation and sequenced on the Illumina NovaSeq 6000 (San Diego, CA, USA) platform.

Nuclei suspensions were stained with 10 μg/mL 7-AAD (Invitrogen, Cat# A1310) and sorted on a BD Fusion Flow Cytometer (BD FACSMelody4-Way Cell Sorter) with a 100 µm nozzle. Nuclei were counted using a Countstar Automated Cell Counter (Countstar Rigel S5) and approximately 20,000 nuclei per sample were loaded into the 10x Chromium system using the Single-Cell 3′ Reagent Kit v3.1 according to the manufacturer’s instructions (10x Genomics). GEM-Reverse Transcription was performed with a thermal cycler using the following program: 53℃ for 45 min, 85℃ for 5 min, and held at 4℃. After reverse transcription and cell barcoding, emulsions were lysed and cDNA was isolated and purified with Cleanup Mix containing DynaBeads and SPRIselect reagent (Thermo Scientific), followed by PCR amplification. The cDNA was then evaluated using an Agilent Bioanalyzer. Enzymatic fragmentation and size selection were applied to optimize the cDNA amplicon size. Then, the P5, P7, i7, and i5 sample indexes and TruSeq Read 2 (read 2 primer sequence) were added by end repair, A-tailing, adaptor ligation, and PCR. The final libraries containing the P5 and P7 primers were sequenced by an Illumina NovaSeq 6000 sequencer with 150 bp paired-end reads, aiming for a coverage of approximately 50,000 raw reads per nucleus.

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.

Mononuclear cells were isolated for sequencing as described above with 3–5 ventral midbrains pooled per sample and sorted for CD45⁺ live cells on a BD FACsAria. Sorted cells were loaded onto the 10X Chromium platform (10X Genomics), and libraries constructed using the Single Cell 3′ Reagent Kit V3.1 according to the manufacturer’s instructions. At least three biological replicates for each group were processed separately (PBS [n = 3], monomer [n = 3], PFF + vehicle [n = 4], PFF + AZD1480 [n = 5]). Samples were sequenced at an average depth of 20,000 reads per cell using Illumina NextSeq 500. Raw base call files were demultiplexed into FASTQ files. Sequencing files were processed and mapped to mm10, and count matrices were extracted using the Cell Ranger Single Cell Software (v 7.1.0) (42 ).

The single‐cell suspensions were used for snRNA‐seq library construction with the Single Cell 3′ Reagent Kit v3.1 (10× Genomics) according to the manufacturer's instructions. The constructed libraries were sequenced on the Illumina HiseqXTEN platform.

The single-cell library was prepared using a commercially available droplet method, the Chromium System from 10× Genomics, Inc. (Pleasanton, CA, USA), and a Single Cell 3′ v3 Reagent Kit according to the manufacturer’s protocol. Sequencing reads were mapped to the GRCh38 reference genome, and bioinformatics processing of the scRNA-seq data was performed using R packages. The detailed methods, including scRNA-seq, bioinformatic analyses, copy number alteration (CNA) detection, and cell–cell communication analysis, are available in the Supplementary Methods.

Cells were loaded for an expected recovery of 10,000 cells per channel. The chip loaded with single-cell suspension was placed on a 10x Genomics Chromium Controller Instrument (10× Genomics, Pleasanton, CA, USA) to generate single-cell droplets containing uniquely barcoded GEMs (Gel Bead-In Emulsions). Single-cell RNA-seq libraries were obtained following the 10x Genomics recommended protocol, using the reagents included in the Chromium Single-Cell 3′ v3 Reagent Kit. The libraries were sequenced on the NovaSeq S2 flow cell (Illumina, 100 cycles).