Computational protocol: Mice lacking the chromodomain helicase DNA-binding 5 chromatin remodeler display autism-like characteristics

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Protocol publication

[…] Total RNA was extracted from the frontal cortex brain tissue of naive, 8-week-old male mice matched with same-sex littermates (three wild types; three Chd5−/−). After collection, tissue was rapidly immersed in RNAlater (Qiagen, Valencia, CA, USA) and total RNA was isolated using the RNeasy Mini Kit (Qiagen, 74104). RNA integrity numbers were calculated using a bioanalyzer to be >7 for all samples. Extracted mRNA was purified with poly-T oligo-attached magnetic beads, heat-fragmented, and both strands synthesized and purified. The 3′ ends were then polyadenylated and RNA sequencing (RNA-Seq) libraries were prepared from samples using the Illumina (San Diego, CA, USA) TruSeq protocol, and then barcoded and sequenced using a 50-bp paired-end run on the Illumina HiSeq 2500. Roughly 20 million paired-end reads were generated for each run. Raw RNA-Seq data were uploaded to the Minnesota Supercomputer Institute Galaxy server. Quality control of raw data was completed using FastQC, and all samples had base sequence quality values (Phred) >30 with no over-represented sequences. FastQ files were then aligned to the mm10_canonical mouse genome and mapped using Tophat. A list of assembled transcripts for each replicate was then analyzed using Cufflinks to estimate abundances (represented by fragments per kilo bases per million mapped reads) and tested for differential expression. Transcriptional differences between wild-type and Chd5−/− replicates were then analyzed in cummeRbund. The lists of significantly differentially regulated transcripts (P<0.05, false-discovery rate<5%) were analyzed using the Ingenuity Pathway Analysis software (Qiagen). [...] Frontal cortex tissue was dissected from postnatal day (PND) 0 to 1 pups (four per genotype matched from independent litters), dissociated, sparsely transfected with a green fluorescent protein (GFP)-encoded construct and plated on poly-l-lysine-coated glass coverslips (~100 000 cells per 35 mM plate) using plating media from Invitrogen (Carlsbad, CA, USA): MEM Eagle (Invitrogen, #14175095), fetal bovine serum (Invitrogen, #16140071), 20% glucose, sodium pyruvate (Invitrogen, #11360070), glutamine (Invitrogen, #25030081) and penicillin/streptomycin (Invitrogen, #15140122). Two hours after plating, media were replaced with maintenance media (Neurobasal medium (Invitrogen, #21103049), B-27 (Invitrogen, #17504044), 200 mM glutamine (Invitrogen, #25030081) and penicillin/streptomycin (Invitrogen, #15140122)), and half the media was subsequently replenished twice per week. At 12 days in vitro, primary neuronal cultures were fixed with 4% paraformaldehyde/PIPES, HEPES, EGTA, and Magnesium Sulphate Heptahydrate (PHEM)/0.12M sucrose, and then blocked with 3% radioimmunoassay grade bovine serum albumin in 1 × PBS for 1 h at room temperature. Cells were then permeabilized with 0.2% Triton-X in PBS at room temperature and blocked again with 3% radioimmunoassay grade bovine serum albumin in PBS. Cells were stained with a primary anti-MAP2 antibody (1:1000; #ab11268; Abcam, Cambridge, UK) in 1% radioimmunoassay grade bovine serum albumin/PBS overnight at 4 °C, followed by secondary (1:100) and 1 × 4,6-diamidino-2-phenylindole (DAPI) counterstain for 30 min at room temperature. Finally, cells were mounted with DABCO mountant, sealed with nail polish and stored at 4 °C. Fluorescent imaging of cells was performed on a Zeiss Axiovert 200 microscope using a × 20 objective and Micro-Manager microscopy software. Images of 15 randomly selected pyramidal neurons were acquired per culture. Cells were analyzed for dendritic arborization using the ImageJ Sholl Analysis plug-in with a 10 μM radius interval. […]

Pipeline specifications

Software tools ImageJ, Sholl analysis
Application Microscopic phenotype analysis
Organisms Mus musculus