GSM8450397: tarsal plates, 21 months wildtype, replicate 1; Mus musculus; RNA-Seq
The dorsal portion of the eyelids and the surrounding tissues near the tarsal plates were removed using a scalpel. The remaining tarsal plates containing Meibomian glands were digested with 0.2% collagenase A in PBS for 1 hour at 37 °C. The tarsal plates were then rinsed twice with PBS and tissues surrounding the Meibomian glands were removed with fine-tip forceps. Tissues from four littermate mice were pooled together in PBS for each replicate. The Meibomian gland tissue-containing solutions were then transferred into 1.5mL centrifuge tubes using Pasteur pipettes. The tissue solution was centrifuged at 300g/min for 5 minutes, the supernatant was carefully removed, and the tissue pellet was rinsed once with PBS. Isolation of nuclei was performed using the protocol (CG000375 Rev B) from 10X Genomics. RNA-seq libraries were prepared based on the 10X Genomics protocol (CG000315 Rev E) using 10000 nuclei per replicate. Single-cell separation was performed using the Chromium Next GEM Chip G Single Cell Kit. The RNA-seq libraries were sequenced in paired-end mode on an Illumina Novaseq platform with the Novaseq S4 flowcell.