GSM7999840: PL_56_OBS_full; Mus musculus; RNA-Seq
Uterine horns containing embryos were rapidly dissected and embryos were placed on ice in sterile PBS. Brains and placentas were isolated, and fetal forebrain and placenta were diced finely with sterile blade and placed into collagenase A (Millipore-Sigma; 11088793001) digestion solution on ice. Cd11b-positive cells were isolated from both placenta and brain tissue. Samples were serially dissociated into a single cell suspension using hand-flamed Pasteur pipettes, and the resultant suspension was enriched for macrophages and monocytes using a 70%/30% Percoll gradient (Millipore-Sigma; GE17-0891-01). This enriched macrophage/monocyte solution was incubated with human and mouse CD11b microbeads (Miltenyi Biotec; 130-093-634), and cells were further enriched for Cd11b-positive macrophages/monocytes using Miltenyi MACS Separation Columns (LS; 130-042-401) and a Miltenyi QuadroMACS Separator (130-090-976). Fresh CD11b+ cells from biological replicates per diet group were prepared for single-cell RNA sequencing (10X Genomics v3.0 chip). Paired fetal brain and placental samples were collected for 17 mice, giving 34 total samples. Approximately 5,900 cells/sample were sequenced, giving 197,000 sequenced cells with an average depth of approximately 22,000 reads/cell).