GSM4593014: Control ASO; Mus musculus; RNA-Seq
Single-cell suspensions from omental adipose tissue from mouse or human were prepared as follows: for mouse, CD45+ cells were sorted using BD FACSAria III (n=3 mice/group pooled); human CD45+ cells (from n=3 bariatric surgery subjects, as above) were isolated using the Miltenyi Human CD45 MicroBead MS selection kit (Miltenyi, 130-045-801) according the manufacturer's instructions.  Cells were processed using the 10X Genomics Chromium v2 Single Cell 3' RNA-seq (10X Genomics, Pleasanton, CA) and Illumina next generation sequencing technologies (Illumina NextSeq 500 Sequencer) and carried out by the uOttawa StemCore Laboratories (mouse) or the UNGC Sequencing Core at UCLA (human). Input cells were quantified and checked for viability using the Countess II (Thermo Fisher). Suspended cells were loaded on a Chromium controller Single-Cell instrument to generate barcoded single-cell Gel Bead-In-Emulsions (GEMs). GEMs were broken and the barcoded cDNA were amplified using the C1000 Touch Thermal cycler with 96- Well Reaction Module. The amplified barcoded cDNA was then fragmented, A-tailed and ligated with adaptors. Finally, PCR amplification was performed to enable sample indexing and enrichment of the 3′ RNA-Seq libraries. The final libraries were quantified using Thermo Fisher Qubit dsDNA HS Assay kit (Q32854) and the fragment size distribution of the libraries were determined using the AATI Fragment analyzer High Sensitivity NGS kit (DNF-486-0500). Pooled libraries were then sequenced using Illumina Nextseq. All samples were sequenced to an average of >50,000 reads per cell, as per 10x Genomics recommendation. cDNA synthesis and library construction was performed using the 10x Genomics Single Cell 3' Reagent Kit (v2)
GEO
GSM4593014