GSM7360137: MULTI-seq barcode library [Pool_2_MS]; Mus musculus; RNA-Seq
Aortas from two 12-week-old C57BL/6J male mice were dissected out intact and flushed extensively with PBS, before microscopic dissection of surrounding perivascular fat. The dissected aortas were digested with LiberaseTM (50 μg/mL; Roche) for 1 h at 37°C, digests neutralized with Iscove's Modified Dulbecco's Medium supplemented with 10% fetal bovine serum and passed through a 40-µm nylon mesh to remove undigested material, before cell collection by centrifugation. The freshly isolated cells were plated in methylcellulose (MethoCult) in replicate wells of 24-well plate for colony formation. After 14 days, colony forming unit - macrophage (CFU-M) were isolated and disaggregated into single-cell suspensions. The resulting progenitor cells were stained with DAPI, and fluorescence assisted cell sorting (FACS)-purified viable cells used for scRNA-seq. Purified progenitor cells were subjected to MULTI-seq labeling as previously described by McGinnis et al. (2019). Briefly, cells were resuspended in 18 µL of 1x PBS, labeled with a unique 2 µM anchor lipid-modified oligonucleotide (LMO)-Barcoded Oligo complex for 5' on ice. After that, 2 µM co-anchor was added and cells incubated on ice for another 5 min. Labeling was quenched with 1 mL of PBS + 2% BSA and cells from each sample in the run mixed for washing the pool with PBS + 1% BSA (twice); final resuspension was in PBS + 1% BSA. From pooled cells, single cell sequencing libraries were prepared using the Chromium Next GEM single-cell 3' kit (v3.1, 10x Genomics) and Chromium system according to manufacturer's specifications. Paired-end sequencing was performed first, on an Illumina Nextseq High using v2 chemistry and subsequently, to increase read depth, on an Illumina Novaseq 6000 using one lane of an S4 flowcell (Illumina).