GSM3597846: VAM2 ND 6; 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 fat tissue was minced and incubated for 35 min at 37oC under gentle agitation (150rpm) in cRPMI (10% FBS, 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 70μm cell strainer. Although all results shown in this manuscript were obtained following the above extraction method, we also carried out a comparative experiment in which we used 2% FFA-free BSA (Fisher BP9704100) in the digestion and staining buffers instead of FBS. RNAseq results between the two extraction methods were highly similar without displaying a single gene with significant adjusted p-value. (data not shown). 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 HiSeq 2500 using v4 chemistry.
GEO Accession
GSM3597846