GSM9513454: Inflamed_3; Homo sapiens; RNA-Seq
Single-cell RNA-seq experiment was performed by experimental personnel in the laboratory of GENECHEM. The tissues were surgically removed and kept in MACS Tissue Storage Solution (Miltenyi Biotec) until processing. The tissue samples were processed as described below. Briefly, samples were first washed with phosphate-buffered saline (PBS), minced into small pieces (approximately 1mm3) on ice and enzymatically digested with 1 mg/mL collagenase I (Gibco)，1 mg/mL collagenase II，60U/mL Hyaluronidase (Sigma), 10U/mL Liberase (Roche) and 0.02mg/mL DNase I (Roche) for 90 min at 37°C, with agitation . After digestion, samples were sieved through a 100µm and 40µm cell strainer, and centrifuged at 300g for 5 min. After the supernatant was removed, the pelleted cells were suspended in red blood cell lysis buffer (Gibco) to lyse red blood cells. After washing with DPBS containing sample buffer, the cell pellets were re-suspended in DPBS containing sample buffer，stained and counted. BD Rhapsody system was used to capture the transcriptomic information of 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. The cells were lysed in the microwell to hybridize mRNA molecules to barcoded capture oligos on the beads. Beads were collected into a single tube for reverse transcription and ExoI digestion. 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 barcode indicating its cell of origin. Whole transcriptome libraries were prepared using the BD Rhapsody single-cell whole-transcriptome amplification (WTA) workflow including random priming and extension (RPE), RPE amplification PCR and WTA index PCR. The libraries were quantified using a High Sensitivity D1000 ScreenTape (Agilent）and High Sensitivity D1000 Reagents (Agilent）on a 4150 TapeStation System (Agilent) and the Qubit High Sensitivity DNA assay (Thermo Fisher Scientific). Sequencing was performed by illumina sequencer (Illumina, San Diego, CA) on a 150 bp paired-end run.