GSM4743751: UC6I; Homo sapiens; RNA-Seq
Adipose tissue was minced into 3 mm pieces and subjected to collagenase digestion for 50 min at 37°C with continuous rotation. Collagenase buffer consisted of 1X PBS containing calcium and magnesium, 2% bovine serum albumin (BSA; MP Bio, USA), 0.2 mg/mL DNase I (Sigma Aldrich, USA), and 1mg/mL collagenase II (Invitrogen, USA). Following digestion, cells were incubated with 0.01 M EDTA for an additional 10 min then filtered through a 100 µM cell strainer (Fisherbrand, USA) and centrifuged at 1500 rpm for 5 min. The pellet was resuspended in 1X RBC Lysis Buffer (Invitrogen, USA) as per manufacturer's instruction. The samples were centrifuged as above, and the final pellet was converted to a single-cell suspension and frozen for later analysis such that all samples could be run at the same time. The cell-freezing protocol for scRNAseq was developed in conjunction with Bio-Rad Genomics. Briefly, the cell pellet was thoroughly mixed by pipetting up and down 10 times. Cells were counted a total of 4 times for each cell preparation to ensure accuracy of total cell count and viability. Aliquots of 5*10^6 cells/ml were prepared in chilled cryopreservation medium (DMEM + 20% FBS + 10% DMSO) and placed in a 4°C pre-chilled CoolCell FTS30 and placed in a -80C freezer for at least 4 hrs. After 4 hrs the cryovials were transferred to liquid nitrogen for long-term storage. CrF vs. H-MAT. Single-cell libraries of the dissociated SVCs were prepared using the Chromium Single Cell 3ʹ GEM, Library & Gel Bead Kit v3 (10X Genomics) and the Chromium Chip B Single Cell Kit protocols, aiming for recovery of 1200 cells for each sample. Briefly, cells were individually partitioned and encapsulated into subnanoliter oil droplets in the Chromium Controller instrument for cell lysis and barcoded reverse transcription of mRNA, followed by amplification, shearing and Illumina library construction. Single-cell-barcoded cDNA libraries were sequenced on the Illumina NextSeq platform.    CrF vs. CD MAT and UC MAT. Dissociated SVCs were first diluted to 2500 cells/uL in PBS with 0.1% BSA. Cells were individually partitioned and co-encapsulated with barcodes into subnanoliter oil droplets using the ddSEQ single-cell isolator (Bio-Rad, USA) as per manufacturer's instructions in the Illumina Bio-Rad SureCell WTA 3' Library Prep Kit. Following cell isolation, the droplets were transferred to a 96-well PCR plate for cell lysis, barcoding, reverse transcription using a thermal cycler. The droplet emulsion was then disrupted for generation of second strand cDNA, followed by fragmentation, tagging and amplification of cDNA. Single-cell-barcoded cDNA libraries were sequenced on the Illumina NextSeq platform at an average read depth of 175,000 reads/sample.