Facsaria 3 cell sorter b5 r3 v3 system

Brain tissue isolated from postnatal (P4), juvenile (P21) and adult (P60) Pdgfrα:Cre-RCE:loxP-GFP mice was dissociated to single cells with neural (for postnatal) and adult (for juvenile and adult) tissue dissociation kits (P; Miltenyi Biotec) without the red blood cell removal step. Cell suspensions were then filtered with a 30 mm filter (Partec). For OPC removal from OL lineage cells from juvenile and adult brains, CD140a (anti-mouse CD140 APC conjugated, BD Bioscience) labelling was performed. GFP+ cells from postnatal brains and GFP+/CD140a- cells from juvenile and adult brains were isolated with fluorescence-activated cell sorting (FACS) using a BD FACSAria III Cell Sorter B5/R3/V3 system. Sorted cells were collected in Eppendorf tubes, centrifuged at 400g, resuspended in Qiazol and stored at −80°C until RNA extraction/cDNA synthesis.

Dissociated cells were FACS sorted based on fluorescence (RFP+ and EGFP+) either into a tube, prior to running them on the C1-STRT (Dataset A), or directly onto the STRT-seq-2i (Dataset B) platform. For Dataset A, BD FACSAria” III Cell Sorter B5/R3/V3 system was used to collect both fluorescent and non-fluorescent populations from Lhx6^cre::R26R-tdTomato and 5HT3a^EGFP mice, followed by a subsequent manual loading into the C1 chip (Fluidigm system). For the cortico-striatal comparison dataset cortical Pvalb^cre::TdT positive cells were sorted on a FACS ARIA II directly into 3 μL of ice cold ACSF-D with 0.5% BSA in the cell collection chamber of a Fluidigm C1 chip to a final concentration 100-150 cells/μL. The collected cells were processed immediately after FACS on the Fluidigm C1 System according to the C1-STRT protocol. For Dataset B Lhx6^cre::R26R-tdTomato and 5HT3a^EGFP positive cells only were sorted using BD FACSAria II SORP, straight onto the chip array (2400 cells/chip prefilled with 50 nL lysis buffer).