title: GSM6234248: YN3, scRNA-seq; Mus musculus; RNA-Seq
library_construction_protocol: Livers were removed from mice after inferior vena cava perfusion. On washing with DMEM, the livers ware cut up in a dish with scissors. Then the livers were then digested into a single cell suspension using liver dissociation kit (Miltenyi, 130-105-807), referring to the script provided by the manufacturer. The cell suspension was filtered through a 70μm strainer and then centrifuged by 300g for 10 minutes at 4℃. Add 70%, 60%, 50%, 40%, 30%, 20% and 10% percoll for 1ml successively to 15ml centrifuge tube, end with the addition of 1ml cell suspension resuspended by DMEM. Next, the tube was centrifuged by 1200g for 15 minutes at 4℃. Extract the layers where 30%, 40% and 50% percoll were located and wash the cells with 1ml DMEM. After counting, 3x10^5 cells were removed and resuspended with 200μl wash buffer (PBS with 2% FBS). The cell samples were blocked by 1μg CD16/32 for 10 minutes at room temperature. Then 2μl CD11b-BV711 antibody was added. The cell suspensions were mixed and incubated for 20 minutes at room temperature, away from light. After being centrifuged by 300g for 10 minutes at 4℃, the cells were washed with wash buffer once and then resuspended with 500μl wash buffer. CD11b-positive cells were then separated by flow cytometer. Single-cell 3' gene expression libraries for each of the 12 samples were prepared separately following the protocols of the Chromium Single Cell 3' Library & Gel Bead kit V3. Briefly, cells and gel beads containing the poly-T primer sequence connected with cell barcode and UMI (unique molecular identifier) were wrapped by "oil droplets" to form GEM (Gel Bead in Emulsion), in which cells lysed and completed reverse transcription according to the manufacturer's instructions. RNA transcripts from single cells were uniquely barcoded. After reverse transcription, barcoded cDNAs were purified, amplified, end-repaired, and ligated with Illumina adapters as per the manufacturer's protocol to generate a single multiplexed library. All resulting libraries were sequenced on the Illumina Novaseq 6000 platform.