GSM7518425: HC1-67Y-F_scRNA-seq; Homo sapiens; RNA-Seq
The 4mm skin tissues were surgically removed from the distal forearm 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 2.5mg/mL collagenase IV, 0.5mg/mL DNase I, and 2 mg/mL dispase for 1h at 37°C, with agitation. After digestion, samples were sieved through a 70µm cell strainer, and centrifuged at 300g for 5 min. 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 35μm cell strainer. Dissociated single cells were then stained for viability assessment using Calcein-AM (Thermo Fisher Scientific) and Draq7 (BD Biosciences). The single-cell suspension was further enriched with a MACS dead cell removal kit (Miltenyi Biotec) 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). The library for each sample was sequenced by Illumina Nova-6000 (Illumina, San Diego, CA) on a 150 bp paired-end run.