GSM6449898: Wildtype homecage control harvested at ZT hour 5, replicate 3 [snRNA-Seq]; Mus musculus; RNA-Seq
Nuclei were extracted from mouse prefrontal cortical tissue using the Nuclei PURE Prep kit (Millipore Sigma, Burlington MA, USA) with the following modifications. Briefly, Pre-frontal cortex (PFC) tissue samples (~0.035 mg per sample/mouse) were processed in 2mL glass dounce homogenizers (Kimble-Chase, Vineland, NJ USA). Nuclei were purified according to manufacturer's instructions, with all volumes reduced to one quarter the recommended amount. Nuclei were separated by centrifugation at 13,000 rcf for 45 mins at 4oC (Sorvall WX-100, F65L-6 x 13.5 rotor, Thermo Fisher, Waltham, MA USA). Single nuclear RNA-seq libraries were generated using the Chromium Single Cell 3' Solution microfluidics platform (10X Technologies, Pleasonton, CA USA). Single-nuclei libraries were generated from the nuclei suspensions using the 10x Genomics Chromium Controller and Single Cell 3' Reagent Kits v3 Chemistry following manufacturer's instructions. Briefly, we targeted capture of 10,000 single nuclei through generation of gel beads in emulsion (GEMs) which allowed partitioning of an individual nuclei along with a bead containing barcoded oligonucleotides. Reverse transcription and barcoding occurred within this emulsion resulting in transcripts from an individual nucleus having a unique molecular identifier. After barcoding, the emulsion was broken, and the cDNA processed in bulk. The barcoded cDNA was first amplified to generate sufficient mass for library construction and then sample index, P5 and P7 adapters were added for Illumina sequencing. The sizes of 10X cDNA libraries were assessed by Fragment Analyzer with the High Sensitivity NGS Fragment Analysis Kit. The concentrations were measured by StepOnePlus Real-Time PCR System (ThermoFisher Scientific, San Jose, CA) with the KAPA Library Quantification Kit (Kapabiosystems, Wilmington, MA). The libraries were diluted to 2 nM with RSB (10 mM Tris-HCl, pH8.5) and denatured with 0.1 N NaOH.  Three pM libraries were loaded onto NextSeq 500 (Illumina, San Diego, CA) for sequencing using the NextSeq 500/550 High Output Kit v2.5 (150 Cycles) . The libraries were sequenced from both ends with 28+8+0+91 cycles (red length 100 bp).