GSM6615651: wildtype fetal brain; Mus musculus; RNA-Seq
Single nuclei from fetal brain were isolated using Pure Prep Nuclei Isolation kit (Sigma, St. Louis, MO, USA). Briefly, frozen brain samples after thawing were minced into small pieces and added to the lysis buffer. Using a dounce homogenizer, the sample were be homogenized till the solution looks evenly mixed, which generally requires 15-20 dounces. A 70µm cell strainer was be used to filter the nuclei from the lysed cells, diluted and layered over a freshly prepared 1.8M sucrose cushion solution to collect single nuclei. After centrifugation and suspension with the kit provided storage buffer (ice cold), final purification of single nuclei was be performed by filtering through a 40µm cell strainer. The purified nuclei were then counted using a Countess II FL Automated Cell Counter (ThermoFisher). The freshly prepared nuclei were used to prepare sequencing libraries using 10X Genomics Chromium Single Cell 3ʹ GEM, Library & Gel Bead Kit v3.1 at the University of Missouri Genomics Technology Core. The nuclei suspension, reverse transcription master mix, and partitioning oil were loaded on a Chromium Next GEM G chip with a capture target of 10,000 nuclei per sample.  Post-Chromium controller GEMs were transferred to a PCR strip tube and reverse transcription performed on an Applied Biosystems Veriti thermal cycler at 53°C for 45 minutes.  cDNA was amplified for 12 cycles and purified using Axygen AxyPrep MagPCR Clean-up beads.  cDNA fragmentation, end-repair, A-tailing, and ligation of sequencing adaptors was performed according to manufacturer specifications.  The final library was quantified with the Qubit HS DNA kit and the fragment size was analyzed using an Agilent Fragment Analyzer system.  Libraries were pooled and sequenced on an Illumina NovaSeq to generate 50,000 reads per cell with a sequencing configuration of 28 base pair (bp) on read1 and 98 bp on read2. Each library was sequenced to a depth of 20,000 paired-end (single-indexing) reads per nucleus using NovaSeq 6000.