title: GSM4680683: Total CD45+ Leukocytes from Ldlr-/- Aorta; Mus musculus; RNA-Seq
library_construction_protocol: The thoracic aorta was carefully isolated, and the perivascular fat was removed. Then the aortic tissue was cut into 2–5 mm pieces and incubated at 37°C for 70 min with gentle shaking in enzyme mixture containing DNase I (90 U/ml), collagenase I (675 U/ml), collagenase XI (187.5 U/ml), hyaluronidase (90 U/ml). Live CD45+ cells were sorted out using BD FACSAriaIII. The cells are resuspended in final concentration of 1000cells/µL in 1X PBS containing 0.04% BSA to minimize cell loss and aggregation. ~17,400 cells are partitioned into nanoliter-scale Gel Bead-In-EMulsions (GEMs) to achieve single cell resolution for a maximum recovery of 10,000 individual cells per sample. Utilizing the v2 Chromium™ Single Cell 3' Library Kit and Chromium instrument (10x Genomics), poly-adenylated mRNA from an individual cell is tagged with a unique 16bp 10x barcode and 10bp Unique Molecular Identifier. Full length cDNA is then amplified to generate sufficient mass for library construction. Enzymatic fragmentation and size selection are used to optimize the cDNA amplicon size (~400bp) for library construction. The final library is sequence ready and contains four unique sample indexes. The concentration of the 10x single cell library will be accurately determined through qPCR (Kapa Biosystems) to produce cluster counts appropriate for the HiSeq4000 platform (Illumina). 26x98nt read lengths are generated targeting 50,000 reads per cell.
attributes.tag: GEO Accession
attributes.value: GSM4680683