GSM7730869: blood monocytes, LDLR-/- ApoB100/100, chow diet; Mus musculus; RNA-Seq
Mice were sacrificed and tissue collections performed as described below. WAT: 200 mg of eWAT was minced and added to 2.5 ul of Miltenyi Adipose Tissue Dissociation solution (supplemented with BSA and HEPES) and incubated 45 min at 37C. During dissociation, the tissue was triturated 3 times (at 25, 35 and 45 min) to reduce cell multiplets. Tissue samples were passed through a 30 um cell strainer and washed with 3 ml of RPMI. Cells were centrifuged at 400 g for 8 min at 4C. The resulting cell pellet was resuspended in 300 ul of FACSs buffer. Samples were lysed using 1X RBC Lysis Buffer, Multi-species (eBioscience #00-4300-54) followed by TotalSeq staining (BioLegend) according to the manufacturer recommendations. Specifically, cells were gently mixed with 9x ice cold 1X RBC lysis buffer and incubated for 3 min on ice. Next, we added PBS to normalize the buffer and collected the cells by centrifugation at 400 g for 8 min at 4C). Cell pellets were resuspended with TotalSeq Mouse Hashtag Ab-DNA conjugate in FACS buffer (PBS-BSA). The solution was incubated in ice for 15-20 min to allow for hashtag Ab binding. Afterwards, cells were washed two times with PBS-BSA to removed unbound hashtag Ab. Dead cells were removed with Miltenyi Dead Cell Removal (Miltenyi Biotec #130-090-101). Blood: 150 ul of EDTA blood were gently mixed with 9x ice-cold 1x red blood cell lysis buffer and incubated for 3 min. Subsequently, cells were centrifuged at 500 g for 5 min at 4C. Cell pellets were resuspended with TotalSeq Mouse Hashtag Ab-DNA conjugate in FACS buffer (PBS-BSA). Next, cells were passed through a 30 um cell strainer and enriched for monocytes by anti-mouse Cd115 magnetic selection with Miltenyi mouse Cd115 MicroBead kit (# 130-096-354) as described by the manufacturer. First, we use 90 ul of cells resuspended in PBS with 0.5% BSAA and 2mM EDTA and mix it with 10 ul of Cd115-Biotin conjugates. After mixing and incubating for 10 min at 4C, cells were washed by adding 1-2 ml of buffer and centrifuged at 300g for 5-10 min at 4C. Cell pellets were resuspended in 80 ul of buffer. 20 ul of Anti-Biotin MicroBeads were added to the solution, mixed and incubated for 15 min at 4C. Cells were washed with 1-2 ml of buffer and centrifuged at 300g for 5-10 min at 4C. Cells were resuspended in 500 ul of buffer. MS columns were place in the magnet and cells were passed through a 30 um cell strainer before adding it to the MS column. All the steps were done at 4C afterwards. The columns were washed three times with 500 ul of buffer. After removing the column from the magnet, we added 1 ml of elution buffer and centrifuged the cells at at 300g for 5-10 min at 4C. From both tissues, cell pellets were resuspended in PBS containing 0.04 % BSA and counted by hemocytometry with trypan blue staining. Cell viability was between 74 % and 85 %. We aimed for a cell concentration of 3000 cells per sample and loaded the cell suspension into the Chromium Controller microfluidics chip (10x Genomics). In each experiment group, cell isolation was carried out separately from 3 male mice and subsequently the cells were pooled in equal proportions into one lane of the Chromium Controller microfluidics chip (10x Genomics). The Chromium Single Cell 3' Kit (v2 Chemistry; 10x Genomics) was used to prepare scRNA-Seq libraries.