NextSeq 500 paired end sequencing; Single-cell RNA-seq of de-differentiated mammary adipocytes during lactation
Single-cell RNA-seq of de-differentiated mammary adipocytes during lactation
Mice were on doxycycline food (600mg/kg chow) for 7 days (10-11 weeks old). SVF fractions from the mammary gland were obtained from female mice. Briefly, dissected mammary glands were digested for 1 hour at 37 degree in PBS containing 10 mM CaCl2, 2.4 units/mL dispase II (Roche Diagnostics Corporation, Indianapolis, IN) and 1.5 units/mL collagenase D (Roche Diagnostics Corporation, Indianapolis, IN). Digested cell/tissue mixture was filtered through a 100 µm cell strainer to remove undigested tissues. The flow-through cells was centrifuged for 5 minutes at 600xg at 4ºC. Cell pellet was resuspended in complete SVF culture medium (DMEM/F12 Invitrogen, Carlsbad, CA; plus Glutamax, Pen/Strep, and 10% FBS) and then filtered through a 40 µM cell strainer to remove clumps and large adipocytes. Samples of SVF cells from the mammary glands (subcutaneous adipose tissue) were first incubated on ice for 20 minutes in 200 uL of 2% FBS/PBS containing anti-mouse CD16/CD32 Fc Block (clone 2.4G2) (1:200). Cells were then incubated with primary antibody (anti-CD31 clone 390 1:200, anti-CD45 clone 30-F11 1:200, CD140a clone APA5 1:100) and were incubated rotating at 4°C for 30 minutes. They were then washed three times with 2% FBS/PBS, and either analyzed using a FACSCantoIITM flow cytometer or sorted by a FACSAriaTM flow cytometer (UT Southwestern Medical Center Flow Cytometry Core Facility). For sorting, cells were initially selected by size, on the basis of forward scatter (FSC) and side scatter (SSC). Live cells were then gated on both SSC and FSC Width singlets, ensuring that individual cells were analyzed. SVF cells isolated from wild-type mice, along with fluorescent-minus-one (FMO) controls, were used to determine background fluorescence levels. All flow cytometry antibodies were rat-derived and conjugated to APC or PE. All antibodies were obtained commercially from BioLegend (San Diego, CA USA). The single-cell RNA-seq was performed using the Smart-seq2 method (Picelli et al., 2013), with several modifications . Briefly, single cells were sorted by FACS into 96-well PCR plate with lysis buffer (2 ul of 0.2% TritonX-100, 1 ul of dNTP (10 uM each), 1 ul of 10 uM biotin-oligo-dT30VN oligo, 0.1 ul of 40U/ul Rnase Inhibitor (Clontech), 0.1 ul of ERCC RNA Spike-In Mix (1:106 diluted, ThermoFisher)). The single cell lysates were then incubated at 72 °C for 3 min and put on ice immediately.  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).