Agilent 4200

Specimens were collected from Wenshan (Yunnan) and Wangmo (Guizhou) between 2019 and 2023. All individuals were maintained on moist soil in the laboratory before RNA extraction and chromosome preparation (animal handling and experiments were approved by the Ethics Committee at Guizhou Normal University, Permission number: 20,230,300,015). The experiments adhered to the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines [36 (link)]. All methods were conducted in compliance with relevant guidelines and regulations.
The specimens were euthanized using ethyl acetate and then preserved in 70% alcohol. For maximum mRNA extraction, five representative organs (brain, heart, liver, skin, and muscle) were collected and mixed from two healthy adult females. Total RNA was extracted from the mixed tissues using TRIzol reagent (Invitrogen, MA, USA) on dry ice, following the manufacturer’s instructions. DNA was removed using TURBO DNase I (Promega, Beijing, China). RNA degradation and contamination were assessed by 1% agarose gel electrophoresis. RNA purity was determined using a NanoDrop 2000 microspectrophotometer (Thermo Scientific, USA; NanoDrop 2000 detection blank reference: DEPC water). The RNA integrity (RIN) was accurately measured with an Agilent 4200 (Agilent Technologies). Only RNA samples with a RIN ≥ 8 were considered suitable for cDNA library construction.

Single‐cell RNA sequencing (scRNA‐seq) was performed in cooperation with CapitalBio Technology Inc. (Beijing, China). Briefly, bone marrow tissues were flushed from adult mouse femurs with PBS buffer and passed through 40 μm strainers, and the LSK cells were collected and counted using a flow cytometer (BD FACS Aria II). Then single‐cell RNA‐seq libraries were constructed according to the instructions accompanying the single cell 3′ Library and Gel Bead Kit V3 (10× Genomics, 1,000,075). The cDNA libraries were subsequently generated, amplified, and assessed for quality control using the Agilent 4200, and single‐cell RNA sequencing was further performed on the Illumina Novaseq6000 sequencer. Read pre‐processing was performed using the 10 × Genomics workflow. For quality control, we set the threshold and removed cells with more than 10% of reads mapping to mitochondrial genes (regarded as low‐quality cells that exhibit extensive mitochondrial contamination). A final dataset of 12,814 LSK cells was preserved to map the landscape of HSC development.
LPG cells were enriched and processed as described above. ScRNA‐seq data of 12,814 LSK cells merged with 19,608 LPG cells were computed and visualized using R packages.

The Chromium Single Cell 3′ Library and Gel Bead Kit V2 (10x Genomics, 120237) and Single Cell A Chip Kit (10x Genomics, 120236) were used for cell capture. The cell suspension (300–600 living cells per microlitre, as determined by Count Star) was loaded onto the Chromium Single Cell Controller (10x Genomics) to generate single-cell gel beads in emulsion (GEMs) according to the manufacturer’s protocol. In short, single cells were suspended in PBS containing 0.04% BSA. Approximately 3173 cells were added to each channel, and the target cell recovery was estimated to be ~16,544 cells in total. Captured cells were lysed, and the released RNA was barcoded through reverse transcription in individual GEMs. Reverse transcription was performed on a S1000TM Touch Thermal Cycler (Bio Rad) at 53 °C for 45 min, followed by 85 °C for 5 min and a hold at 4 °C. cDNA was generated and then amplified, and quality was assessed using an Agilent 4200 (performed by CapitalBio Technology, Beijing).