title: GSM8262107: obob, rosi, iWAT, biological replicate  1, scRNAseq; Mus musculus; RNA-Seq
library_construction_protocol: 300mg of the white adipose tissue (eWAT or iWAT) were collected (small biopsies from several locations of the fat depot to represent tissue environment), cut into small pieces, suspended in phosphate-buffered saline (PBS) (calcium, magnesium free) (GIBCO) containing 2mg/mL of type II collagenase (Worthington), and digested for 40 minutes at 37°C with agitation (100 rpm). The resulting dissociated tissue was resuspended in DMEM (GIBCO) with 10% FBS, gently mixed, passed through 100μm strainers, and centrifuged at 150g for 8 min at 4C. Next, supernatant and floating adipocytes were removed, and pellet was resuspended in DMEM (GIBCO) with 10% FBS and centrifuged at 150g for 8 min at 4C. Red blood cells were lysed using ACK lysis buffer (GIBCO). Lysate pellets were first washed with DMEM (GIBCO) with 10% FBS, centrifuged at 150g for 8 min at 4C and then PBS with 0.04% BSA (Gemini). The resulting pellets were finally resuspended in 200uL of PBS, 0.04% BSA (Gemini), passed through 40μm tip-strainers, counted, and further used for 10X 3'GEX library preparation and sequencing. The Chromium Single Cell Gene Expression Solution upgrades short read sequencers to deliver a scalable microfluidic platform for 3ʹ digital gene expression by profiling 500-10,000 individual cells per sample. A pool of ~3,500,000 10x Barcodes are sampled separately to index each cell's transcriptome. It is done by partitioning thousands of cells into nanoliter-scale Gel Beads-in-emulsion (GEMs), where all generated cDNA shares a common 10x Barcode. Libraries are generated and sequenced from the cDNA and 10x Barcodes are used to associate individual reads back to the individual partitions. In addition to the poly(dT) primer that enables the production of barcoded, full-length cDNA from poly-adenylated mRNA, the Single Cell 3ʹ v3.1 Gel Beads also include two additional primer sequences (Capture Sequence 1 and Capture Sequence 2), that enable capture and priming of Feature Barcoding technology compatible targets or analytes of interest. Only the poly(dT) primers are used in this protocol for generating Single Cell 3ʹ Gene Expression libraries. GEMs are generated by combining barcoded Single Cell 3ʹ v3.1 Gel Beads, a Master Mix containing cells, and Partitioning Oil onto Chromium Next GEM Chip G. To achieve single-cell resolution, cells are delivered at a limiting dilution, such that the majority (~90-99%) of generated GEMs contain no cell, while the remainder largely contains a single cell. Immediately following GEM generation, the Gel Bead is dissolved, primers are released, and any co-partitioned cell is lysed. Primer containing (1) an Illumina TruSeq Read 1 (read 1 sequencing primer), (2) 10x Barcode, (3) 12nt unique molecular identifier (UMI), and (3) 30nt poly(dT) sequence are mixed with the cell lysate and a Master Mix containing reverse transcription (RT) reagents. Incubation of the GEMs produces barcoded, full-length cDNA from poly-adenylated mRNA. After incubation, GEMs are broken, and pooled fractions are recovered. Silane magnetic beads are used to purify the first-strand cDNA from the post GEM-RT reaction mixture, which includes leftover biochemical reagents and primers. Barcoded, full-length cDNA is amplified via PCR to generate sufficient mass for library construction. Enzymatic fragmentation and size selection are used to optimize the cDNA amplicon size. TruSeq Read 1 (read 1 primer sequence) is added to the molecules during GEM incubation. P5, P7, a sample index, and TruSeq Read 2 (read 2 primer sequence) are added via End Repair, A-tailing, Adaptor Ligation, and PCR. The final libraries contain the P5 and P7 primers used in Illumina bridge amplification. A Chromium Single Cell 3' Gene Expression library comprises standard Illumina paired-end constructs which begin and end with P5 and P7. The 10x Barcode and 12 bp UMI are encoded in Read 1, while Read 2 is used to sequence the cDNA fragment. Sample index sequences are incorporated as the sample index read. TruSeq Read 1 and TruSeq Read 2 are standard Illumina sequencing primer sites used in paired-end sequencing. These libraries were sequenced using Illumina's NovaSeq6000 platform in paired end 2x100bp configuration.