Single cell sequencing of de-differentiated dermal adipocytes

Purpose: The goals of this study are to compare the de-differentiated dermal adipocytes with normal skin fibroblasts. Methods: Library prepared followed by 10X Genomics standard protocol. Transcriptome was generated by high throughput sequencing. Overall design: We previously derived a doxycycline-inducible (Tet-On) lineage-tracing model that allows for the indelible labeling of Adiponectin-expressing adipocytes (Adiponectin-rtTA; TRE-Cre; Rosa26RmT/mG; herein, Adipose Chaser mice). Prior to exposing animals to doxycycline, all cells express membrane tdTomato from the Rosa26 locus. Dermal adipose shows high plasticity. They display dynamic changes in volume and cell numbers in synchrony with hair cycling. But, no obvious apoptosis was reported during the recession of dermal adipose tissue. We set out to test the hypothesis that whether dermal adipose tissue undergo de-differentiation during the recession of dermal adipose tissue, and the difference between de-differentiated adipocytes and normal skin fibroblasts. Following the exposure to doxycycline-containing chow diet, Cre-mediated excision of the loxP-flanked tdTomato cassette occurs in Adiponectin-expressing (P28-P35) cells, and membrane-bound GFP (mGFP) expression is constitutively activated in adipocytes. Therefore, we labeled the pre-existing dermal adipocytes with GFP following doxycycline-containing chow diet during the second anagen when dermal fat have the most large volume. Next, we collected single cell suspension from skin at the pro-longed second telogen when dermal adipose tissue have most strong recession (P56-P63). FACS sorted CD31-CD45-Pdgfra+GFP- and CD31-CD45-Pdgfra+GFP+ cells were collected. CD31-CD45-Pdgfra+GFP- and CD31-CD45-Pdgfra+GFP+ cells were mixed together with the ratio of 1:1, approximately 7000 cells per group. To this end, we performed single cell RNA-sequencing of CD31-CD45-Pdgfra+GFP- and CD31-CD45-Pdgfra+GFP+ cells.