GSM8833066: Myt1l heterozygous male mouse 10; Mus musculus; RNA-Seq
Mice were anesthetized and brains were quickly extracted, meninges removed, and the hypothalamus was dissected using 3D printed molds to allow for precise extraction of tuberal hypothalamus from A/P -0.5 mm to -2.6 mm, M/L -1.5mm and 1.5mm and DV of 4-6mm. Dissected hypothalamus were flash frozen in liquid nitrogen, and stored at -80°C. Molds will be made available on Tinkercad. Nuclei were isolated from flash frozen tissue. Samples were processed in 9 batches of 4, counterbalanced for sex and genotype. Brain tissues were Dounce homogenized in ice-cold homogenization buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 1 mM DTT, 1X complete EDTA-free Protease Inhibitor (Roche 4693132001), and 0.2U/ul RNasin Inhibitor (Promega N2515)) with a 2 ml KIMBLE KONTES Dounce Tissue Grinder (DWK 885300-002) with 15 strokes with the “A” large clear ance and 15 strokes of the “B” small clearance pestle. The homogenate was transferred to a 15 ml LoBind Eppendorf centrifuge tube (Eppendorf 0030122208). Walls of the homogenizer tubes were washed with 1 ml of ice-cold homogenization buffer and combined with the homogenate. Nuclei were pelleted by centrifugation in a swinging bucket rotor (Eppendorf S-4-104) at 500 × g for 5 mins at 4 °C. Pellets were resuspended in ice-cold 1 ml homogenization buffer. Density gradient centrifugation was performed to purify the nuclei from debris and myelin from tissue dissociation. An ice-cold 50% iodixanol-PBS working solution was prepared by diluting the stock solution of 60% w/v Optiprep (Sigma-Aldrich D1556) with DPBS (ThermoFisher 14190136). A 35% (w/v) iodixanol solution was then made by diluting the 50% iodixanol with DPBS. To make the 25% Iodixanol layer, 1 ml ice-cold 50% iodixanol was added to 1 ml of the resuspended pellets from the first centrifugation. This was carefully layered on top of 2 ml 35% iodixanol in a clear polycarbonate tube (Beckman Coulter 355672) and centrifuged at 10,000 × g for 30 min at 4 °C in a S0410 swinging bucket rotor (Beckman Coulter 364660) with no braking using an Allegra 64R (Beckman Coulter 367586). After centrifugation, myelin and debris at the top of the 25% iodixanol layer were aspirated and discarded. Up to 2 mL of purified nuclei from the interface of the 25% and 35% iodixanol layers were collected using a low retention P1000 pipette and transferred to a clean 15 ml LoBind Eppendorf centrifuge tube. The volume was brought up to 6 ml with nuclei wash and resuspension buffer (1% bovine serum albumin (Sigma A1595), 0.2 U/uL RNase Inhibitor (Promega N2515), in calcium- and magnesium-free DPBS) and pelleted by centrifuging at 500 × g for 5 min at 4 °C. Supernatant was carefully removed and discarded, while the pellet was washed with nuclei wash buffer and centrifuged at 500 × g for 5 min at 4 °C to ensure removal of carryover iodixanol. The pellet was then resuspended in 1x ice-cold DPBS, filtered through a 40 μm Flowmi cell strainer, and manually counted using a hemocytometer with propidium iodide. 200 k–1.7 M nuclei were used as input for the ScaleBio Sample Fixation Kit (Scale Biosciences 2020001) according to the manufacturer's low sample input protocol. After fixation, the nuclei were manually counted once more and checked for quality using a microscope with a 60× objective. The nuclei were then stored at −80 °C until all samples had been collected and fixed. Libraries were prepared from cohort 1 (10 male WT, 10 male Hets, 10 female WTs, 6 female Hets). Frozen fixed nuclei were thawed on ice and counted twice via hemocytometer. Using the ScaleBio Single Cell RNA Sequencing Kit v1.0 (Scale Biosciences 2020008), all 36 samples were prepared in one batch. Nuclei were loaded at 10,000 per well (2-3 wells/sample) to add RT barcodes as sample identifiers. Pooled nuclei were distributed across the 384-well plate for Ligation Barcode addition, then pooled again with 1600 nuclei per well for second strand synthesis. After tagmentation and indexing PCR, libraries were pooled (5μl each), cleaned with 0.8X SPRIselect beads (Beckman Coulter B23317), and fragment size/concentration quantified using High Sensitivity D5000 Screentape (Agilent) and NEBNext Library Quant Kit (New England Biolabs E7630S). Sequencing was performed on a Novaseq X Plus (Illumina; shared 25B flowcell) to a target depth of 20,000 reads per nucleus.