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| Status |
Public on May 22, 2014 |
| Title |
MGE_WT_1hrsKCl |
| Sample type |
SRA |
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| Source name |
MGE 10 DIV
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| Organism |
Mus musculus |
| Characteristics |
cell type: E14 MGE derived neurons, 10 DIV
treatment: 55mM KCL
time: 1 h
genetic background: C57Bl/6
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| Treatment protocol |
At DIV 9, the cells were quieted O.N. with TTX and AP-5 and membrane-depolarized on DIV 10 for 0, 1 and 6 hours with 55 mM extracellular KCl
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| Growth protocol |
Mouse E14 MGE- and CTX-derived neurons were cultured for 10 days
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted with Trizol according to the manufacturer's instructions
Whole Transcriptome sequencing (WT-Seq, RNA-Seq: sequencing of total RNA): WT-Seq was performed according to a protocol/kit now available from Life Technologies, with minor modifications that are included below. Briefly, 5ug of total RNA isolated from DIV10 mouse MGE-cultures cultures was depleted of ribosomal RNAs using two rounds of Human/Mouse RiboMinus treatment (Life Technologies) with overnight ethanol precipitations for sample re-concentration. The removal of ribosomal RNAs was confirmed on a Bioanalyzer Nano Chip (Agilent). 500–1000ng of riboRNA-depleted total RNA was fragmented by heating for 18 minutes at 95°. Fragmentation was followed by size selection of ~50 to ~150bp fragments using the flashPAGE denaturing PAGE-fractionator (Life Technologies) and ethanol precipitation overnight. The resulting RNA was directionally ligated, reverse-transcribed, and RNAseH treated. After trial PCR to assess library quality and quantity, 30ul cDNA was run on a native 6% PAGE gel. The 90–120bp size window (corresponding to 50–80bp RNA insert size) was cut from the gel, shredded, and inserted directly into a 400ul PCR reaction using standard WT-Seq kit components and submitted to 11–15 cycles of PCR. The PCR product was phenol-chloroform extracted, ethanol precipitated, and resuspended in 20ul WT-Seq gel loading buffer. The resulting sample was run on a 6% native PAGE gel, and the 150–175bp size range (corresponding to 60–85bp) was cut from the gel, shredded, and extracted overnight in WT-Seq PAGE elution buffer. The resulting library was filtered through 0.45um spin filters (Life Technologies) to remove gel pieces and ethanol precipitated. We note that WT-Seq can detect neither the 5'-most fragment from transcripts with 5'-modified ends (such as mRNA 5' 7-methyl-guanosine caps) nor the 3'-most fragment from transcripts with 3'-modified ends. However, for transcripts long enough to produce multiple 50+ bp fragments, WT-Seq should detect the remaining fragments.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
AB SOLiD System 3.0 |
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| Data processing |
Target sequences for alignment of 35-bp reads included (1) 21 mm9 autosomal and sex chromosomes plus (2) a single pseudochromosome containing a read-length-dependent splice library (~2.2M sequences, each 35-68bp). The latter comprised all possible minimal intragenic sequences of two or more exons (based on the RefSeq annotation) for which one or more exon-exon junctions could be crossed by reads of the required length.
SOLiD raw reads were aligned using Corona with 0-3 colorspace (cs) mismatches for 35-bp reads. Uniquely mapped reads output to SOLiD-format "unique.csfasta.gz.ma" files served as input for further processing. SOLiD quality scores are in the .qual.gz files.
Aligned reads were minimally filtered for QC, removing any reads with uncalled bases or with unmapped or nonuniquely mapped sequences.
Employing "MAPtoFeatures" perl scripts, the loci of uniquely mapped reads were compared to the loci of all genic features (exons, introns, UTRs, CDSs, etc., and all junctions between them) of all genes based on the RefSeq annotation. Every overlap of an individual read with genic features, including exon-exon splice junctions, was counted towards the features' "nrds" while the number of bases overlapped was added to their "rdbp". For every genic feature the average Density (read coverage per bp) was calculated, equal to rdbp per feature length. Each sample's Densities were renormalized to a standard total of 10M reads (with the standard read length of 35bp), and further corrected for mappability as described below. With these conventions, units of Density always equal RPKM times 0.35. Normalized Densities were treated as expression levels comparable among all samples. Also note that in analyses using the MAPtoFeatures code, a "gene" is a construct based on the Refseq annotation of transcripts: all exons from all transcripts assigned to a given gene are unioned together; the resulting discontinuous pieces are labeled as individual exons ("EXN"); and the gaps between them are defined as the gene's introns ("INT").
Exonic features (EXN), for each gene's whole exonic region and for individual exons, were corrected for mappability as follows. A collection of fake reads was constructed from 35-bp sequences beginning at every single base of the mapping targets, i.e., the mm9 reference genome and its accompanying 35-bp splice library pseudochromosome. These were aligned as for any other set of reads, allowing up to 3 mismatches, and only uniquely mapped ones were considered further. The mappability index of an exon or an exonic region was defined in such a way that perfect coverage -- unique mapping of every fake read -- yields an index of unity; without correcting for normalization, this case would correspond to Density = 35. The mappability index "i" for a genomic region is thus i = <unnormalized Density of fake reads>/<read length>. Densities for a region are corrected for mappability by dividing the uncorrected (but normalized) Density for that region by its index i. (Occasionally, double-counting of splice library members in multiple transcripts for the same gene lead to indices slightly greater than one.)
Genome_build: Mouse mm9 (NCBI reference assembly C57BL/6J, build 37, version 1, July 2007).
Supplementary_files_format_and_content: The "Genes-FEATURES-READS" .tsv files are spreadsheets output by MAPtoFeatures. Each row has data for one gene (with NCBI Entrez Gene IDs). A 1-line header labels 14 columns containing genomic information, followed by blocks of columns labeled "num" (number of each feature type per gene), "bp" (total length of each feature type per gene, followed by data columns. Data in the remaining blocks are distinguished by reads that mapped S[ense] or A[ntisense] to each gene: "nrds" (number of overlapping reads wholly contained within each feature type per gene), "rdbp" (total number of bases of reads that overlap each feature type at all, including reads crossing feature junctions), "Density" (expression levels, equal to normalized, mappability-corrected coverage as described above). Some columns labeled "-ALL" give concatenated values for all individual features, listed 5' to 3', within each gene. Exon-exon junctions, labeled "SPL", are notated as <count>(<exons>) with "^" between adjoining exons; e.g., "2(5^6)" indicates 2 reads crossing the junctions between exons 5 and 6, and "1(3^5^6)" for 1 read crossing from exon 3 to 5, through all of 5, and crossing to exon 6. Exon-intron junctions, labeled "JXN", are notated as, e.g., "1(8>8<)" for 1 read crossing from exon 8 into intron 8 or "3(8<9>)" for 3 reads crossing intron 8 and exon 9. Exonic mappability indices are listed (column "S_mappability", applicable to both strands) where appropriate as "EXN=<i0>[<i1>|<i2>|...]", where <i1>, <i2>, ... are the indices for individual exons, and <i0> is the index calculated over all exonic bases. (Owing to a glitch that was later corrected, the 3'-most exon's index is missing from the output, though not from the exonic Density calculation, for genes with multiple exons.)
Supplementary_files_format_and_content: The "cross_reads_and_features__LOG" .txt files document each MAPtoFeatures analysis, including total reads used for normalization, totals per feature across the genome, and other statistics.
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| Submission date |
Mar 05, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Ivo Spiegel |
| E-mail(s) |
ivo_spiegel@hms.harvard.edu
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| Organization name |
Harvard Medical School
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| Department |
Department of Neurobiology
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| Lab |
Michael E. Greenberg
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| Street address |
220 Longwood Ave
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| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
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| Platform ID |
GPL9318 |
| Series (2) |
| GSE55590 |
Identification of activity-induced genes in cortical inhibitory neurons (RNA-Seq) |
| GSE55591 |
Identification of activity-induced Npas4-regulated genes in cortical inhibitory and excitatory neurons |
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| Relations |
| BioSample |
SAMN02673320 |
| SRA |
SRX481194 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1339893_Ivo_MGE-KCl_1hr_Genes-FEATURES-READS-r35_x500_N10M_ALL-GENES_EXN-ALL_INT-ALL_SPL_JXN_END_mm3_MPX.tsv.gz |
8.5 Mb |
(ftp)(http) |
TSV |
| GSM1339893_cross_reads_and_features_LOG_100114_163740_O_Ivo_MGE-KCl_1hr.txt.gz |
20.4 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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