title: GSM8673812: H-1; Homo sapiens; RNA-Seq
library_construction_protocol: Single-cell RNA-seq experiment was performed by experimental personnel in the laboratory of NovelBio Co.,Ltd. The tissues were removed and kept in MACS Tissue Storage Solution (Miltenyi Biotec) until processing. The tissue samples were processed as described below. Briefly, samples were first digested using a mixture of 0.5 mg/ml Collagenase I (Thermo Fisher)and 1 mg/ml Neutral Protease (Thermo Fisher)for a total of 25 minutes at 37℃, which was divided into two stages: an initial digestion period of 10 minutes followed by a second digestion period of 15 minutes. Due to an insufficient number of cells observed after the initial digestion, an additional digestion step was performed using 0.1% trypsin for 15 minutes at 37℃. Throughout the digestion process, agitation was applied to ensure efficient enzymatic action.After digestion, samples were sieved through a 70 μm cell strainer, and centrifuged at 400×g for 5 min. After the supernatant was removed, the pelleted cells were suspended in red blood cell lysis buffer (Miltenyi Biotec) to lyse red blood cells. After washing with PBS containing 0.04% BSA, the cell pellets were re-suspended in PBS containing 0.04% BSA and re-filtered through a 35 μm cell strainer. Dissociated single cells were then stained with AO/PI for viability assessment using Countstar Fluorescence Cell Analyzer. BD Rhapsody system was used to capture the transcriptomic information of the single cells. Single-cell capture was achieved by random distribution of a single-cell suspension across >200,000 microwells through a limited dilution approach. Beads with oligonucleotide barcodes were added to saturation so that a bead was paired with a cell in a microwell. Cell-lysis buffer was added so that poly-adenylated RNA molecules hybridized to the beads. Beads were collected into a single tube for reverse transcription. Upon cDNA synthesis, each cDNA molecule was tagged on the 5′ end (that is, the 3′ end of a mRNA transcript) with a unique molecular identifier (UMI) and cell label indicating its cell of origin. Whole transcriptome libraries were prepared using the BD Rhapsody single-cell whole-transcriptome amplification workflow. In brief, second strand cDNA was synthesized, followed by ligation of the WTA adaptor for universal amplification. Eighteen cycles of PCR were used to amplify the adaptor-ligated cDNA products. Sequencing libraries were prepared using random priming PCR of the whole-transcriptome amplification products to enrich the 3′ end of the transcripts linked with the cell label and UMI. Sequencing libraries were quantified using a High Sensitivity DNA chip (Agilent) on a Bioanalyzer 2200 and the Qubit High Sensitivity DNA assay (Thermo Fisher Scientific). All libraries were sequenced by illumina sequencer (Illumina, San Diego, CA) on a 150 bp paired-end run.