GSM5082472: KO_2; Mus musculus; RNA-Seq
For preparation of RNA-seq samples, Itgav(+);CD45(−) meibomian gland cells from WT and Hsd3b6−/− mice were obtained by FACS. We used four mice per replicate, with two replicates for both genotypes. Tarsal plates from four mice were pooled into HBSS containing 0.25% Trypsin and 50 µg/mL DNaseI, incubated for 15 min at 37℃, and minced. Then, digested tissues were dissociated in PBS containing 20% FBS and 0.5 mM EDTA and filtered through 70-µm strainer. Cell were resuspended into ice-cold PBS containing 2% FBS, 0.5 mM EDTA, and 50 µg/mL BSA) and further incubated with anti‐CD45-APC/Cyanine7 (Biolegend) and anti‐Itgav-PE (Biolegend). Cell sorting was performed using a FACSAria II Flow cytometer with FACSDiva software (BD Biosciences). Itgav(+);CD45(−) meibomian gland cells (approximately 10,000 cells per replicate) were sorted into DMEM/Ham's F12 medium containing 10% FBS. FACS-purified meibomian cells were pelleted and directly lysed into RLT buffer (Qiagen) containing 1% 2-mercaptoethanol. Total RNA was extracted using an Rneasy micro kit (Qiagen). Total RNA was amplified using a SMARTer Ultra Low Input RNA Kit for Sequencing (Clontech). Then RNA-seq libraries were prepared using a NEBNext Ultra II RNA Library Prep Kit for Illumina (NEB) according to the manufacturers' protocols.
GEO Accession
GSM5082472