GSM9084505: Cortical nuclei, 2 pooled samples, 1 male het/ 1 female wt, biol rep 1; Mus musculus; RNA-Seq
Frozen issue from the first 6 samples was processed into nuclei using 2mL glass dounce homogenizers containing 2mL of EZ Prep lysis buffer each. Tissue for 3 samples was dounced over ice, 25 times with pestle A and 35 times with pestle B. Each solution was transferred to a 15 mL conical tube, then the same douncing process was repeated for the second set of 3 samples. Solutions from the latter 3 samples were pooled with those of the first 3 such that each pool contained one male and one female of opposite genotype. An additional 6 mL of EZ Prep lysis buffer was added to each conical tube and the tubes were incubated on ice for 5 minutes. Homogenates were centrifuged at 500xg for 5 minutes at 4°C, the supernatant was discarded, and the pellet was washed twice in NSB (1X PBS with 0.01% BSA and 0.1% RNAse inhibitor). The final pellet was resuspended in 1mL NSB then passed through a 20uM cell strainer. Nuclei were counted using a LUNA-FL cell counter and Acridine Orange/ Propidium Iodide stain. The 10X Genomics nuclei isolation kit, with protocol as described, was used to isolate nuclei for the final 4 samples. These samples were combined into 2 pools, each containing one male and one female of opposite genotype, then nuclei were counted as described previously. Nuclei suspensions had concentrations adjusted to target 10,000 nuclei for sequencing per pool, then used as input for the 10X Genomics 3' Gene Expression Assay. Library preparation was performed according to the manufacturer's instructions (Next GEM Single Cell 3' Gene Expression v3.1 protocol, 10X Genomics). Briefly, nuclei were resuspended in a reverse transcription master mix and loaded into a Chromium Next GEM Chip G along with barcoded gel beads and partitioning oil. The chip was run on a Chromium controller to generate Gel Beads-in-Emulsion (GEMs). Within each GEM, reverse transcription occurred, producing cDNA tagged with a cell barcode, UMI, and an Illumina Read 1 primer sequence. GEMs were then broken and the barcoded cDNA was purified using Dynabeads, amplified by PCR, and cleaned using SPRIselect beads. During library construction, cDNA was fragmented, end-repaired, A-tailed, and ligated to sequencing adapters that include the Illumina Read 2 primer binding site. After adapter ligation and SPRIselect cleanup, sample index PCR was performed to incorporate P5 and P7 sequences and sample indices, followed by a final SPRIselect cleanup. Libraries were QCed and sequenced on an Agilent BioAnalyzer, batches 1, 2, and 4 were sequenced on an Illumina NovaSeq 6000 and batches 5 and 6 were sequenced on an Element Biosciences AVITI.