GSM5655409: p32; Sus scrofa; RNA-Seq
The brain cell nuclei were isolated according to our previous protocol (Li et al., 2018; Zhu et al., 2018) with some modifications. Hippocampal regions (DG, CA1, CA2-4, Sub) and adjacent entorhinal cortex were dissected from three frozen adult human brains (Table S1). In order to avoid experimental bias and evenly dissociate the tissue for cell nuclei isolation, whole tissue was finely pulverized to powder in liquid nitrogen with mortar and pestle (Coorstek #60316, #60317). All buffers were ice cold and all reagents used for consequent nuclear isolation were molecular biology grade unless stated otherwise. 5 - 10 mg of pulverized tissue was added into 5 ml of ice-cold lysis buffer consisting of 320 mM sucrose (Sigma #S0389), 5 mM CaCl2 (Sigma #21115), 3 mM Mg(Ace)2 (Sigma #63052), 10mM Tris-HCl (pH 8) (AmericanBio #AB14043), protease inhibitors w/o EDTA (Roche #11836170001), 0.1 mM EDTA (AmericanBio #AB00502), RNAse inhibitor (80U/ml) (Roche #03335402001), 1mM DTT (Sigma #43186), and  0.1% TX-100 (v/v) (Sigma#T8787). DTT, RNAse Protector, protease inhibitors, and TX-100 were added immediately before use. The suspension was transferred to Dounce tissue grinder (15ml volume, Wheaton #357544; autoclaved, RNAse free, ice-cold) and homogenized with loose and tight pestles, 30 cycles each, with constant pressure and without introduction of air. The homogenate was strained through 40 um tube top cell strainer (Corning #352340) which was pre-wetted with 1ml wash buffer: (250 mM sucrose (Sigma #S0389), 25 mM KCl (Sigma #60142), 5mM MgCl2 (Sigma #M1028), 20mM Tris-HCl (pH 7.5) (AmericanBio #AB14043; Sigma #T2413), protease inhibitors w/o EDTA (Roche #11836170001), RNAse inhibitor (80U/ml) (Roche #03335402001), 1mM DTT (Sigma #43186)). Additional 4 ml of wash buffer was added to wash the strainer. Final 10 ml of solution was mixed with 10 ml of 50% Optiprep (Axis-Shield# 1114542) solution (50% iodixanol (v/v), 250 mM sucrose (Sigma #S0389), 25 mM KCl (Sigma #60142), 5mM MgCl2 (Sigma #M1028), 20mM Tris-HCl (pH 7.5) (AmericanBio #AB14043; Sigma #T2413), protease inhibitors w/o EDTA (Roche #11836170001), RNAse inhibitor (80U/ml) (Roche #03335402001), 1mM DTT (Sigma #43186)) by inverting the tube 10x and carefully pipetted into 2 centrifuge tubes (Corning #430791). The tubes were centrifuged at 1000g, for 30 min at 4 ˚C on centrifuge (Eppendorf #5804R) and rotor (Eppendorf #S-4-72). Upon end of centrifugation, the supernatant was carefully and completely removed and total of 5 ml of resuspension buffer (250 mM sucrose (Sigma #S0389), 25 mM KCl (Sigma #60142), 5mM MgCl2 (Sigma #M1028), 20mM Tris-HCl (pH 7.5) (AmericanBio #AB14043; Sigma #T2413), protease inhibitors w/o EDTA (Roche #11836170001), RNAse inhibitor (80U/ml) (Roche #03335402001), 1mM DTT (Sigma #43186)) was added carefully on the pellets in tubes and centrifuged at 1000g, for 10 min at 4 ˚C on the same centrifuge and rotor. Supernatants were then carefully and completely removed, pellets were gently dissolved by adding 100 ul of resuspension buffer (see above) and pipetting 30x with 1ml pipette tip, pooled and filtered through 35 um tube top cell strainer (Corning #352340). Finally, nuclei were counted on hemocytometer and diluted to 1 million/ml with sample-run buffer: 0.1% BSA (Gemini Bio-Products #700-106P), RNAse inhibitor (80U/ml) (Roche#03335402001), 1mM DTT (Sigma #43186) in DPBS (Gibco #14190). 10X Chromium Single Cell 3ʹ v3