GSM6213081: Pilo1; Mus musculus; RNA-Seq
GSM6213081
INTACT protocol was modified from Mo et al (2015). Briefly, mice were euthanised by cervical dislocation and the brains were harvested and placed in ice-cold homogenisation buffer (HB; 0.25 M sucrose, 25 mM KCl, 5 mM MgCl2, 20 mM tricine, pH 7.8) supplemented with cOmplete™, EDTA-free protease inhibitor cocktail (Roche). Forebrains were isolated and homogenised in a 5 mL Potter-Elvehjem tissue grinder with 10 strokes of the pestle (Corning). 0.3% IGEPAL®-CA 630 (Sigma) in PBS was then added to the homogenate and further homogenised with another 5 strokes of the pestle. Homogenates were then filtered through a 40 µm cell strainer (Falcon) and mixed 1:1 with 50% OptiPrep™ Density Gradient Medium in dilution buffer (DB, 150 mM KCl, 30 mM MgCl2, 120 mM tricine, pH 7.8) before being layered over an iodixanol gradient composed of 30% and 40% OptiPrep™ in HB. Samples were centrifuged at 10,000 g for 18 min with SW28 swinging bucket rotor at a deceleration setting of 5 (Beckman Coulter). After centrifugation, nuclei were extracted from the interface between 30% and 40% of the OptiPrep™ gradient. To immunoprecipitate SUN1-sfGFP positive nuclei by INTACT, immunoprecipitation buffer (IPB, 0.25 M sucrose, 25 mM KCl, 5 mM MgCl2, 20 mM tricine, 0.4% IGEPAL®-CA 630, pH 7.8) was added to nuclear extracts (approximately 4-5 million nuclei) with 3 µg of anti-IgG or anti-GFP antibodies used for each reaction and incubated for 30 min at 4°C. Pre-washed Dynabeads™ Protein G (Invitrogen) were added for another 40 min before nuclei were isolated with extensive and repeated binding steps to enhance binding efficiency. Dynabeads™-bound nuclei were pelleted and washed with IPB, then lysed in 1 mL of TRIzol™ Reagent (Invitrogen) and stored at -80°C until RNA extraction. Total RNA was extracted then column-purified with PicoPure™ RNA isolation kit (Applied Biosystems™) according to manufacturer's instructions. Directional poly(A) mRNA libraries were prepared for RNAseq by NovogeneAIT Genomics (Singapore) and 45 M 150 bp paired-end reads per sample were generated by sequencing on the Illumina Novaseq 6000 platform.