NextSeq 500 sequencing; Single-cell RNA-seq of de-differentiated mammary adipocytes during lactation
Single-cell RNA-seq of de-differentiated mammary adipocytes during lactation
The brown preadipocyte IBA cells were cultured in DMEM supplemented with 10% FBS in 5% CO2. The cells were subjected to adipogenic differentiation by adding DMI cocktail (Dexamethasone, IBMX and Insulin) 2 day post confluence. As the challenging of isolating single mature adipocyte, the differentiating single cells (2 days after the initiating of differentiation) were harvested instead. Then the cells were subjected to scRNA-seq together with the sorted SVF cells.  RT mix (0.06 ul of 1M MgCl2, 2 ul of 2M Betaine, 0.1 ul of Biotin-TSO-LNA oligo, 0.25 ul of Rnase Inhibitor, 2 ul of 5* Maxima H- RT buffer, 0.1 ul of Maxima H Minus Reverse Transcriptase (ThermoFisher), 1.39 ul of H2O) was added to each well, and the samples were subjected to reverse transcription. The cDNAs were subsequently amplified with PCR mix (12.5 ul 2* KAPA HiFi HotStart ReadyMix (KAPA), 0.25 ul of 10 uM Biotin-ISPCR oligo, 2.25 ul of H2O).  The amplified cDNAs were then purified, tagmentated by in-house Tn5 (Picelli et al., 2014) and subsequently amplified using Nextera i5 and i7 Adapters (Illumina).