title: GSM7051580: WT, replicate 2, scRNAseq; Mus musculus; RNA-Seq
library_name: GSM7051580
library_construction_protocol: SVF was isolated from the subcutaneous white adipose depots of 8-week old male wild-typeC57Bl/6J mice. Briefly, the adipose depots were dissected and then manually minced in DMEM/F12 and digested with collagenase 2 (1.5 units/ml; Roche). They were then incubated at 37°Cwith agitation for 60 min and vortexing for 10s every 10-15mins. HBSS with 2% FBS was addedand the dissociated cells were centrifuged at 350 × g for 5 min. Floating mature lipid-filledadipocytes were aspirated and the cell pellets were resuspended in ACK lysis buffer to removethe red blood cells, followed by the addition of HBSS with 2% FBS. The cells were then filteredthrough a 40-mM filter and pelleted by centrifugation at 350 × g for 5 min. Cell pellets wereresuspended in 200ul FACS staining buffer (5-10 x 105 cells/tube) and incubated with thefollowing antibodies for 30 min at 4°C protected from light. Cells were washed twice with FACSbuffer to remove unbound antibodies. The cells were sorted using a BD FACS Aria cell sorter (BD Biosciences). Cellss were washed in HBSS-/- and resuspended in HBSS-/- supplemented with 0.04% w/v BSA. Library was performed according to the manufacter's instructions (single cell 3' v2 protocol, 10x Genomics). Briefly, GCs were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.