GSM9667736: Denerv_951; Mus musculus; RNA-Seq
Brains were excised excluding the medulla, without dissecting meninges and choroid plexus, and manually chopped (0.5–1.0 mm2 in size), before enzymatic dissociation using 0.4mg/ml Collagenase D, 2mM HEPES (BI Industries), 10μg/ml DNase (Sigma), and 2% FCS for 20 min at 37°C. For density gradient separation, the pellet was resuspended with 37% Percoll in PBS and centrifuged at 800g for 20 min at 4°C with no brake; the supernatant was then discarded. Cells were suspended in ice-cold sorting buffer (PBS supplemented with 2  mM  EDTA and 2% FCS) supplemented with anti-mouse CD16/32 (1:100, no. 101302,  BioLegend) to block Fc receptors before labeling with fluorescent antibodies against cell-surface epitopes (Supplementary Table 1). For scRNA sequencing, samples were gated for CD45+ CD11b+ after exclusion of debris and doublets, and sorted using an ARIA-III instrument (BD  Biosciences), and analyzed with BD FACSDiva (BD Biosciences) software. Isolated single cells were sorted into 1.5ml Eppendorf tubes containing PBS enriched with 10% ultra-pure BSA and then immediately subjected to library preparation for scRNA sequencing. n single-cell RNA libraries were prepared using 10x Chromium Next Gem Single cell 3' reagent kit (version 3.1) according to the manufacturer's instructions