GSM4103298: SVF, 34-week CHOW rep4; Mus musculus; RNA-Seq
Dissociation media was prepared by adding 2 grams of bovine serum albumin (BSA) to 100 mL of 4.5g/L glucose DMEM media (Invitrogen) and warmed in a 37C water bath to dissolve. Adipose tissue was dissected at the termination of the study. The fat pad was rinsed with PBS before being gently minced in dissociation media with scissors and a razor blade. The minced tissue was then digested by transferring to a 50 mL falcon tube containing 25 mL of 1 mg/mL type I collagenase (Worthington) dissolved in dissociation media and placed in a 37C water bath with gentle shaking for approximately 40 minutes. The dispersed tissue was then passed through a 100 µm mesh filter and centrifuged at 200xg for 10 minutes. The floating adipocytes were carefully removed and added to a tube containing approximately 5 volumes of trizol (Qiagen) and immediately snap frozen in a bath of LN2. The pelleted SVF was resuspended in erythrocyte lysing buffer (155 mM NH4Cl, 10 mM KHCO3, 1 mM EDTA), incubated at room temperature for 3 minutes and then centrifuged at 200xg for 10 minutes. Pelleted SVF was resuspended in 2 mL trizol (Qiagen) and immediately snap frozen in a bath of LN2. Cell fractions were stored at -80C. cDNA libraries for RNA-seq analysis were prepared from total RNA (isolated as described above) according to Illumina TruSeq stranded mRNA sample prep LS protocol. Pooled sample libraries were sequenced on Nextseq 500 instrument with Nextseq 500/550 High output kit, 150 cycles (Illumina).