Drop-Seq (single-cell RNA-Seq) of human stromal vascular fraction of subcutaneous adipose tissue: Sample Drop-Seq_Hs_SAT_SVF_238-1B-1
Whole tissue subcutaneous adipose specimens were freshly collected from the operating room. Skin was removed, and adipose tissue was cut into 1- to 2-inch pieces and rinsed thoroughly with 37C PBS to remove blood. Cleaned adipose tissue pieces were quickly minced in an electric grinder with 3/16-inch hole plate, and 400 ml of sample was placed in a 2-l wide-mouthed Erlenmeyer culture flask with 100 ml of freshly prepared blendzyme (Roche Liberase TM, research grade, cat. no. 05401127001, in PBS, at a ratio of 6.25 mg per 50 ml) and shaken in a 37C shaking incubator at 120 r.p.m. for 15–20 min to digest until the sample appeared uniform. Digestion was stopped with 100 ml of freshly made KRB (5.5 mM glucose, 137 mM NaCl, 15 mM HEPES, 5 mM KCl, 1.25 mM CaCl2, 0.44 mM KH2PO4, 0.34 mM Na2HPO4 and 0.8 mM MgSO4), supplemented with 2% BSA. Digested tissue was filtered through a 300 μM sieve and washed with KRB/albumin and flow through until only connective tissue remained. Samples were centrifuged at 233g for 5 min at room temperature, clear lipid was later removed, and floated adipocyte supernatant was collected, divided into aliquots and flash-frozen in liquid nitrogen. After removal of floated adipocytes, remaining supernatant was aspirated and the remaining pelleted stromal vascular fraction (SVF)was combined from multiple tubes. The combined SVF was washed 2 times with 50ml cold PBS with 233g for 5 min centrifugation between washes. Erythrocytes were depleted with two rounds of 25 ml. ACK lysing buffer (Gibco™ A1049201) exposure (5 minutes at RT followed by 233g x 5 min centrifugation). Remaining SVF pellet was further washed x 2 with 50ml cold PBS prior to counting on hematocytometer and loading onto Drop-seq microfluidic devices. Drop-seq was performed as described (Macosko et. al., 2015), with flow rates of 2.1 mL/h were used for each aqueous suspension and 12 mL/h for the oil. Libraries were sequenced on the Illumina NextSeq 500, using between 1.6-1.7 pM in a volume of 1.2 mL HT1 and 3 mL of 0.3 μM Read1CustSeqB (GCCTGTCCGCGGAAGCAGTGGTATCAACGCAGAGTAC) using 20 x 8 x 60 read structure. Drop-Seq (scRNA-Seq)