title: GSM9107467: Single_Cell_multi-omics_seq_GEX_baseline-Control_Jarid2_KO_Male_KSL_Cells; Mus musculus; OTHER
library_name: GSM9107467
library_construction_protocol: For Single Cell RNA Seq experiment: C-kit+ cells were enriched from the bone marrow of 18-week post-transplant recipient mice (5 × 10⁵ CD45.2/CD45.1 whole bone marrow primary transplant). Donor-derived (CD45.2) KSL (c-Kit+ Sca1+ Lineage–) cells were subsequently sorted from the enriched population and resuspended at a concentration of 1,200 cells/μL in PBS supplemented with 0.04% BSA. For Single Cell CITE Seq Experiment, C-kit+ cells were enriched from bone marrow of untransplanted control, Jarid2Δ/Δ, and Ezh2Δ/Δ mice. Following enrichment, c-Kit+ Sca1+ Lineage– (KSL) cells were subsequently sorted by FACS (Beckman Coulter MoFlo). Cells were first stained with biotin-conjugated anti-Sca1 (Ly-6A/E) antibody (Supp Table 2) for 20 minutes at 4 °C, then washed and incubated with a panel of six TotalSeq™-B barcoded antibodies (BioLegend) targeting surface markers: streptavidin-PE (Sca-1), CD117, CD48, CD150, CD135, and CD201 (Supp Table 3). Antibody staining was performed using 1 µL of each antibody in 50 µL of PBS containing 0.04% BSA, for 30 minutes on ice, protected from light. Following staining, cells were washed with cold PBS containing 0.04% BSA, filtered through a 40 µm cell strainer and assessed for viability (>90%) using Trypan Blue exclusion. After that, cells were resuspended at 1,200 cells/μL in PBS + 0.04% BSA. Single-cell RNA Seq libraries were prepared using the Chromium Next GEM Single Cell 3′ Reagent Kits v3.1 (Dual Index) (10x Genomics, PN-1000268), following the manufacturer's protocol (User Guide CG000317 Rev D) with minor modifications. Cell partitioning and barcoding were performed using the Chromium Next GEM Chip G Single Cell Kit (PN- 1000127) and Chromium Dual Index Kit TT Set A (PN-1000215). GEM generation and reverse transcription (GEM-RT), followed by post-GEM cleanup, were conducted according to standard procedures. Purified cDNA was amplified for 11–13 cycles based on input cell number and quality, and then cleaned using SPRIselect beads (Beckman Coulter, Cat. No. B23318). Amplified cDNA was assessed using the Agilent Bioanalyzer High Sensitivity DNA Kit (Agilent, Cat. No. 5067-4626) to determine concentration and size distribution. Final gene expression (GEX) libraries were constructed as per the 10x Genomics protocol, adjusting the number of amplification cycles according to cDNA input. The libraries were quantified using the KAPA Library Quantification Kit for Illumina platforms (Roche/KAPA Biosystems, Cat. No. KK4824) and sequenced on the Illumina NovaSeq X Plus system using the XP workflow on an S4 Flow Cell with a paired-end 28 × 10 × 10 × 150 bp sequencing strategy. A target median depth of 50,000 reads per cell was achieved. Single-cell transcriptome and protein profiling were performed using the Chromium Next GEM Single Cell 3′ Reagent Kits v3.1 (Dual Index) (PN-1000268) in conjunction with the Feature Barcode Kit for Cell Surface Protein (PN-1000262), following the manufacturer's protocol (User Guide CG000317 Rev D) with slight optimizations to the staining and preparation steps. Cells were loaded onto the Chromium Controller using the Chromium Next GEM Chip G (PN-1000127), targeting recovery of approximately 10,000 cells per channel. Gel bead-in-emulsion (GEM) generation, barcoding, and reverse transcription were carried out using the recommended thermal cycling conditions: Lid temperature 53 °C, 53 °C for 45 min, and 85 °C for 5 min; hold at 4 °C. Post-RT GEMs were broken, and barcoded cDNA was purified using Dynabeads™ MyOne™ Silane (PN-2000048). Amplified cDNA containing both mRNA- and antibody-derived tags (ADTs) was generated via PCR using cycling conditions recommended by 10x Genomics and cleaned up using SPRIselect reagent (Beckman Coulter, Cat. No. B23318). Separate libraries were prepared for gene expression and Feature Barcode (protein) profiling: 1. Gene Expression Library: A portion of the cDNA was enzymatically fragmented, end-repaired, A-tailed, and ligated to adapters, followed by sample index PCR amplification using the Dual index Kit TT Set A (PN-1000215). 2. Feature Barcode Library: A separate cDNA aliquot was amplified using primers targeting the Feature Barcode constructs using Dual index Kit NT Set A (PN-1000242). Final libraries were quantified using a Qubit 4 Fluorometer and assessed for size distribution using the Agilent Bioanalyzer High Sensitivity DNA Kit (Cat. No. 5067-4626). Libraries were pooled in appropriate ratios and sequenced on the Illumina NovaSeq X Plus system using an S4 flow cell with the following read configuration: 28 bp (Read 1), 10 bp (i7 index), 10 bp (i5 index), and 90 bp (Read 2). A target median sequencing depth of ~50,000 reads per cell was used for gene expression and 5,000–10,000 reads per cell for antibody-derived tags.