GSM8832611: Healthy microbiome, 2 wks old, mouse 3; Mus musculus; RNA-Seq
When the pups reached two weeks of age, we collected whole-brain samples from four of the full-term microbiome mice (three female, one male) and four of the preterm microbiome mice (two female, two male). To isolate brain cells, we used the Neural Tissue Dissociation Kit - Postnatal Neurons created by Miltenyi Biotec. We followed the protocol established by Miltenyi Biotec for use of the kit without small modifications: using a 40 µm strainer instead of a 70 µm one, incubation after addition of enzyme mix 1 for eight minutes instead of fifteen, and incubation after dissociation of tissue for six minutes instead of ten. The change in filter size was necessary to remove debris, and we shortened the incubation times to reduce potential cell stress and RNA degradation. We prepared scRNA-seq libraries using Chromium Next GEM Single Cell 3′ v.3.1 reagent kit (10x Genomics, CA, USA). We barcoded cells using the Chromium Controller (10x Genomics). Eleven polymerase chain reaction cycles were applied to generate cDNA and thirteen cycles for final library generation. The final scRNA-seq libraries were sequenced with the Illumina NovaSeq X (Illumina, CA, USA).