RNA-seq of mesenchymal cells from young and aged mouse submandibular glands (C57BL/6)
Five C57BL/6 mice (8w, male) were pooled to form Young group and five C57BL/6 mice (18m, male) were pooled as Age group. Mesenchymal cells of MGs were enriched by fluorescence-activated cell sorting (FACS) using negative selection for epithelial cells (EpCAM+) before sequencing (sorting details were described in 2.5 section). Total RNA was extracted from sorted cells using the Trizol reagent kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. RNA integrity was verified by Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). Sequencing libraries were prepared with the TruSeq Stranded mRNA Library Prep Kit (Illumina, 20020594) following the manufacturer's protocol. Libraries were quantified, pooled, and sequenced on an Illumina NovaSeq6000. Raw sequencing reads were quality-trimmed using fastp (version 0.18.0) and aligned to the mouse reference genome (GRCm39) using HISAT2.2.4. For each transcription region, a fragment per kilobase of transcript per million mapped reads (FPKM) value was calculated to quantify its expression abundance and variations, using RSEM software.