title: GSM8171686: CD326-negative from skin cells_cDNA; Mus musculus; OTHER
library_construction_protocol: The ear skin was minced finely with scissors and resuspended in 2 ml of Accutase followed by incubation in a shaking incubator for 10 min at 37°C. The digests were centrifuged at 800 g for 3 min and the cell pellets were resuspended in Liberase solution consisting of 250 µg/ml Liberase TM and 2,000 units/ml DNase I in RPMI-1640 supplemented with 10 mM HEPES and 5% (v/v) fetal bovine serum followed by incubation in a shaking incubator for 30 min at 37°C. Procedure for the digestion with Liberase and DNase was repeated twice. The resulting cells were isolated by 25% (v/v) Percoll density gradient, and then filtered through a 40-µm cell strainer. The cells were isolated CD326-negative fraction by autoMACS. Preparation of the scRNA-seq library was performed as described previously (Shichino et al. Commun Biol 5, 602, 2022). In brief, cells prepared from mouse skin were stained with BD Ms Single-Cell Multiplexing Kit (anti-mouse CD45, 30-F11) or Custom BD Single-Cell Multiplexing Set (anti-mouse MHC-H2 class I, M1/42). Obtained single-cell suspensions were subjected to a BD Rhapsody system with Targeted mRNA and AbSeq Amplification Kit following the manufacture's instructions. Resultant beads were reverse transcribed, treated with exonuclease I, and subjected to TAS-Seq workflow for cDNA and hashtag library amplification. Ligand products were purified and performed barcording PCR. Size distribution of amplified products was analyzed by a MultiNA system (MCE-202). Resultant libraries and barcorded hastag libraries were quantified using KAPA Library Quantification Kit. Pooled libraries were sequenced on an Illumina NovaSeq 6000 sequencer with an Illumina NovaSeq 6000 S4 Reagent Kit v1.5. Two billion clusters, equivalent to 400 Gb/analysis, were obtained by pair-ended analysis of 100 bp of sequencing data. BD Rhapsody