Mouse brain slicer

Immediately following refeeding, mice were deeply anesthetized with ketamine/xylazine (80mg/kg/ 12mg/kg) then injected with 100 ng of biotin-YVAD-CMK ICV using a microinjection unit attached to a Kopf stereotaxic instrument (Tujunga, CA). Two hours following biotin-YVAD-CMK administration mice were euthanized using CO₂ and perfused with 30 mls of ice cold PBS followed by 30 mls of 4% paraformaldehyde. Brains were sectioned coronally at 1.5 and 3.5 mm from the interaural using a Mouse Brain Slicer (Zivic Instruments, Pittsburgh, PA). Slices were fixed in 4% paraformaldehyde for 24 hours, paraffin embedded and sectioned at 5 μm. Fixed tissues were blocked with Peroxidazed 1 (BioCare, Pacheco, CA), detected with SS horse radish peroxidase (BioCare), stained with liquid 3.3’-diaminobenzidine chromogen (BioCare) and counterstained with hematoxylin (BioCare) utilizing the BioCare IntelliPATH system (BioCare).

Male hemizygous Dlx6a-cre mice (Jax stock #008199) were crossed with female homozygous INTACT mice (flox-Sun1-eGFP, Jax stock #021039) to yield Dlx6a-cre::INTACT offspring for scATAC-seq experiments. Brains from P28 Dlx6a-cre::Sun1-eGFP mice were harvested, sectioned coronally on a mouse brain slicer (Zivic Instruments), and the primary visual cortex was dissected in ice-cold ACSF. Tissue was then transferred to a dounce homogenizer containing Lysis Buffer (10 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl2, 0.01% Tween-20, and 0.01% IGEPAL CA-630, 0.001% Digitonin). Tissue was homogenized with 10 strokes of pestle A, 10 strokes of pestle B, and incubated for 5 min on ice before being filtered through a 30 μm filter and centrifuged at 500xg for 10 min at 4μc. The pellet was resuspended in 1% BSA for sorting GFP+ nuclei on a Sony SH800S cell sorter. Nuclei were sorted into Diluted Nuclei Buffer (10X Genomics). The scATAC library was prepared using the 10x Genomics platform with the Chromium Single Cell ATAC Library & Gel Bead Kit v1.0 (PN-1000111), Chromium Chip E Single Cell kit (PN-1000156) and Chromium i7 Multiplex Kit N, Set A (PN-1000084) as instructed by the manufacturer. High quality data was recovered from approximately 60% of the input nuclei. Libraries were sequenced using a Nova-Seq S2 100 cycle kit (Illumina).

On P45, offspring mice were sacrificed by first administering 5% isoflurane by inhalation for 30 sec followed by cervical dislocation. Subsequently, blood was collected by heart puncture into 1.1ml z-gel serum collection tubes (Sarstedt; Germany). Serum was then collected according to the manufacturer’s instructions and stored frozen in −80 °C until analysis. Brains were macro-dissected using a mouse brain slicer (1mm coronal section slice intervals; Zivic Instruments; Pittsburgh, PA, USA) and sections of the prefrontal cortex and the striatum were collected into RNALater (Thermo; Waltham, MA, USA) and kept frozen in −80 °C until analysis. Intestines were dissected, colon and cecal contents collected separately and flash frozen while the intestinal tissue (~2 cm of the proximal colon and ~2 cm of the terminal ileum) were rinsed in PBS and frozen in RNALater. To control for effects by the time of collection, mice from different groups were sacrificed in an alternated fashion. All samples were then assigned an identification number that prevented from direct identification of the groups to facilitate blinded analysis of samples downstream.