|
| Status |
Public on Dec 31, 2016 |
| Title |
Mecp2_ko_p57_Veh_IP |
| Sample type |
SRA |
| |
|
| Source name |
Hippocampus
|
| Organism |
Mus musculus |
| Characteristics |
age of animal: postnatal day 57
cell-type: Hippocampal CA1 neurons (Cck-EGFP-L10a)
drug treatment: Vehicle
strain: C57/Bl6
|
| Treatment protocol |
Chronic CTEP treatment (0.4 mg/kg) was administered via subcutaneous injection (for mice younger than P30) or oral gavage (for mice older than P30) once every 48 hr. This dose was chosen after a pilot study revealed that the dose of 2 mg/kg used in previous studies in fragile X (Michalon et al., 2012) and 16p11.2 microdeletion (Tian et al., 2015) mouse models caused significant weight loss in the Mecp2 mice.
|
| Growth protocol |
Mecp2 KO mice (Mecp2 tm1.1bird from The Jackson Laboratory, 003890) and WT littermates were maintained on C57/Bl6 background by breeding Mecp2 -/+ females with WT males from Charles Rivers.The Cck-bacTRAP GM39) line was provided by Dr. Nathaniel Heintz at the Rockefeller University. In this mouse, EGFP fused to the ribosomal protein L10a was inserted in the bacterial artificial chromosome (BAC) in such a way that expression was driven by the cholecystokinin (Cck) promoter. All experiments were performed on littermate WT and Mecp2 KO mice, with the experimenter blinded to genotype.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Translating mRNAs were immunoprecipitated from acutely dissected hippocampal tissue and purified as previously described (Heiman et al., 2008, Heiman et al., 2014). RNA samples were analyzed with Bioanalyzer (Agilent) to confirm RNA integrity (RIN>7). RNA concentrations were determined by Quant-it Ribogreen RNA assay (Life Technologies).
Purified RNA (1~1.5 ng) was converted to cDNAs with Ovation RNA-seq V2 system (NuGEN). 500 ng cDNAs from each sample were fragmented to around 200bp size using a Covaris S2 ultrasonicator. Fragmented cDNA (100 ng) was end-repaired, dA-tailed and ligated to adaptors using the NEBNext Ultra DNA library prep kit (New England Biolabs). Sequencing libraries were prepared using the KAPA HiFi Hotstart Ready Mix PCR Kit (Kapa biosystems) and NEBNext Multiplex Oligos for Illumina (Index Primers Set1 and 2). Library quality was examined by Qubit dsDNA HS Assay Kit (Life Technologies) and Bioanalyzer using High Sensitivity DNA kit (Agilent). Final concentrations of sequencing libraries were quantified by qPCR using KAPA Library Quantification Kit (Kapa biosystems). TRAP-seq libraries were sequenced on a HiSeq 2500 or a NextSeq 500 sequencer (Illumina).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
Ribosome-associating mRNA
DESeq2_htseq-counts_Veh.vs.CTEP.txt
|
| Data processing |
Sequencing reads (51bp, single-end) were first mapped to the Mus musculus primary assembly reference genome (Gencode GRCm38/release M6) using STAR-2.4.2a. Uniquely mapped reads that overlapped with exons of known protein coding genes were counted using htseq-count python script. Differential expression was calculated using DESeq2 (Love et al., 2014), and P-values were adjusted for multiple testing using the Benjamini-Hochberg procedure.
Genome_build: GRCm38/release M6
Supplementary_files_format_and_content: tab-delimited text files include DESeq2-normalized count values (dervied from htseq-count script) for each sample.
|
| |
|
| Submission date |
May 31, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Hao Wu |
| E-mail(s) |
haowu7@gmail.com
|
| Phone |
617-713-8660
|
| Organization name |
Harvard Medical School/HHMI
|
| Department |
Genetics
|
| Lab |
Yi Zhang
|
| Street address |
149G Warren Alpert Building, 200 Longwood Avenue
|
| City |
Boston |
| State/province |
Massachusetts |
| ZIP/Postal code |
02115 |
| Country |
USA |
| |
|
| Platform ID |
GPL19057 |
| Series (1) |
| GSE82068 |
Negative allosteric modulation of mGluR5 partially corrects pathophysiology in a mouse model of Rett Syndrome |
|
| Relations |
| BioSample |
SAMN05190575 |
| SRA |
SRX1810447 |
