GSM4475060: VAM1 CMAF 5; Mus musculus; RNA-Seq
To isolate leukocytes from adipose tissue mice were anesthetized with a mixture containing 12.5mg/mL ketamine, 2.5mg/mL xylazine, and 25 mg/mL acepromazine and intracardially perfused with PBS with 5 mM EDTA. After dissection, the eWAT was minced and incubated for 35 min at 37oC under gentle agitation (150rpm) in cRPMI (2% BSA FFA-free [Fisher BP9704100], 1mM of Sodium Pyruvate [Gibco, 11360-070], Glutamax 1x [Gibco, 35050-061], MEM non-essential amino acids [Gibco, 11140-050], Hepes [Gibco, 15630-080], Pen Strep [Gibco, 15140-122] and RPMI [Gibco, 21870-076]) containing 1mg/mL of collagenase type VIII (Sigma-Aldrich, C2139) and 100ug/mL of DNase I (Sigma-Aldrich, 10104159001). After digestion, the suspension was washed with ice-cold cRPMI and spun down. The floating fat layer was removed and the leukocyte-enriched pellet was ressuspended in cRPMI and filtered through a 100 μm cell strainer. Total RNA from sorted target cell populations was isolated using TRIzol LS (Invitrogen; 10296010) followed by DNase I (Qiagen, 79254) treatment and cleanup with RNeasy Plus Micro kit (Qiagen, 74034). RNA quality was assessed using pico bioanalyser chips (Agilent, 5067-1513). Only RNAs displaying a RNA integrity number (RIN) of 9 or higher were used for downstream procedures. RNAseq libraries were prepared using the Nugen Ovation Trio low input RNA Library Systems V2 (Nugen; 0507-08) according to the manufacturer's instructions by the NYU Genome Technology Center. Pooled libraries were sequenced as 50 nucleotide, paired-end reads on an Illumina NovaSeq6000 using v4 chemistry.