title: GSM3666204: MeCP2_KO_whole_cell_RNA_rep5; Mus musculus; RNA-Seq
library_construction_protocol: Forebrain (cortex and hippocampus) was dissected from 10 8-week-old male MeCP2 knock-out and 10 wild-type littermate controls and 10 8-to-12-week-old male R306C and 10 WT littermate controls and flash frozen. Tissue was removed from dry ice and placed directly into a dounce with 5 mL of Homogenization buffer (HB: 250 mM sucrose, 25 mM KCl, 5 mM MgCl2, 20 mM Tricine KOH pH 7.8, 0.04% BSA, 5 µg/mL Actinomycin D, and RNAsin (Promega)). After 10 strokes with the tight pestle, a 5% Igepal (Sigma) solution in HB was added to a final concentration of 0.32%, followed by five additional strokes with the tight pestle. At this point, 200 μL of lysate was pipetted into a tube with 600 µL Trizol LS (Invitrogen) as the “whole cell” sample. The remaining lysate was filtered first through a 40 μm cell strainer and then through a 20 μm Celltrics filter. Lysate was diluted 1:2 in HB to dilute Igepal to 0.16%, then centrifuged at 500 g for 5 minutes at 4°C. The nuclear pellet was washed twice with 1 mL HB by resuspending and centrifuging at 500 g for 5 minutes at 4°C. Nuclei were resuspended in 1mL HB and 200μL was pipetted into a separate tube, spun at 500 g for 5 minutes at 4°C, and the pellet was resuspended in Trizol as the “nuclear fraction”. The remaining 800 μL was spun at 500 g for 5 minutes at 4°C, then resuspended in 200 μL Glycerol buffer (20 mM Tris pH 8, 75 mM NaCl, 0.5 mM EDTA, 50% glycerol, 0.85 mM DTT). 200 μL of Nuclear lysis buffer (1% Igepal, 20 mM Hepes pH 7.5, 300 mM NaCl, 0.2 mM EDTA) was added and nuclei were resuspended, vortexed, and incubated at 4°C for 5 minutes. Chromatin was spun at 16,000 g for 10 minutes at 4°C, and the pellet was resuspended in 800 μL Trizol and homogenized to solubilize. For Dnmt3a cKO mice, visual cortex was dissected and homogenized in Trizol. For all samples, total RNA was chloroform extracted and purified with the Qiagen RNeasy Micro Kit with on-column DNase treatment. RNA-seq libraries were generated with the NEBNext Ultra Directional Library Prep Kit with rRNA depletion.
attributes.tag: GEO Accession
attributes.value: GSM3666204