title: GSM9602257: Sample4 of Run6, Fetal hippocampal tissue section; Homo sapiens; RNA-Seq
library_construction_protocol: Nuclei were isolated from the frozen punched tissue or sections using an optimized isolation protocol. Briefly, samples were homogenized using a glass Dounce homogenizer (15 gentle strokes) in 1 ml of ice-cold Homogenization Buffer (HB), which contained mM sucrose, 5 mM calcium chloride, 3 mM magnesium acetate, 10 mM Tris, 0.1 mM EDTA, 0.1% Igepal, 0.1 mM PMSF, 1 mM 2-mercaptoethanol, and UltraPure water, along with 5 μl RNasin Plus (Promega, Benelux, Belgium). The homogenate was filtered using a 70 μm strainer and washed with 1.65 mL HB, bringing the final volume to 2.65 ml. An additional 2.65 ml of Gradient Medium (containing 5 mM calcium chloride, 50% OptiPrep (Sigma-Aldrich, Belgium), 3 mM magnesium acetate, 10 mM Tris, 0.2 mM PMSF, and 1 mM 2-mercaptoethanol) was added to achieve a final volume of 5.3 ml. To isolate the nuclei, OptiPrep Diluent Medium (ODM) consisting of 150 mM potassium chloride, 30 mM magnesium chloride, 60 mM Tris, and 250 mM sucrose was prepared. The sample was layered onto a 4 ml 29% cushion (29% Optiprep, ODM) using a P1000 pipette, and the weight was balanced with HB. Centrifugation was carried out at 7,700 rpm for 30 min at 4°C using a SW41Ti rotor. After centrifugation, the  supernatant was removed with a plastic Pasteur pipette, and the lower supernatant was aspirated using a P200 pipette. The nuclei were resuspended in 200 μl of resuspension buffer (1x PBS, 1% BSA, and 0.2 U/μl RNasin Plus), transferred to a fresh tube, and washed again with 100-200 μl of the same buffer. The resuspended solution was pooled, and any clumps were disrupted by pipetting with a P200. The sample was then filtered through a 0.35 μm strainer using a Falcon tube. A 9 μl-aliquot of the sample was mixed with 1 μl of propidium iodide (PI) stain, loaded onto a LUNA-FL slide, and left to settle for 30 seconds. The nuclei were examined under a LUNA-FL Automated Cell Counter to assess numbers and morphology. Post cDNA amplification cleanup and construction of sample-indexed libraries and their amplification followed manufacturer's directions (10x Genomics, CG000183_Rev_A), with the amplification step directly dependent on the quantity of input cDNA. In order to reach sequencing saturation of ~65% (~45,000 reads/nucleus), single nucleus libraries were run using paired-end sequencing with single indexing on the NovaSeq6000 platform, following manufacturer's instructions (10x Genomics, CG000204_Rev_A). To avoid lane bias, multiple uniquely indexed samples were mixed and distributed over several lanes.