alias: GSM3678358
title: GSM3678358: AB2576; Mus musculus; RNA-Seq
library_construction_protocol: Mice were euthanized and nd perfused immediately through the left ventricle of the heart with 20 ml of phosphate-buffered saline (PBS) to remove circulating leukocytes from the tissue. The epididymal (visceral) adipose tissue (EAT) was readily located and excised right above the epididymis. The subcutanous adipose tissue (SAT) was obtained from the inguinal fat depot. The tissue was placed in a 50 ml tube with 10 ml DMEM without phenol red at room temperature, cut into tiny bits with scissors, incubated with collagenase II at 37°C for 20 min. while gently agitating, filtered through a 100-μm cell strainer, and spun down at 500g for 10 min with low acceleration/brake, beginning at room temperature and cooling to 4°C. Cells were resuspended in 500 μl RBC lysis solution (Sigma) and incubated on ice for 2-5 min. (depending on the initial amount of tissue) and washed. The resulting cell suspension was incubated with anti-CD45 or a cocktail of antibodies for selected markers. Single-cell RNA-seq libraries were prepared as previously described {Jaitin, 2014}. Briefly, mRNA from single cells sorted into capture plates were barcoded and converted into cDNA and then pooled using an automated pipeline. The pooled sample was linearly amplified by T7 in vitro transcription, and the resulting RNA was fragmented and converted into a sequencing-ready library by tagging the samples with pool barcodes and Illumina sequences during ligation, RT, and PCR. Each pool of cells was tested for library quality and concentration as described previously {Jaitin, 2014};
identifiers.namespace: GEO
identifiers.id: GSM3678358