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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Nov 02, 2016 |
| Title |
Human rep 1 neurons 0hr post-KCl stim |
| Sample type |
SRA |
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| Source name |
ScienCell
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| Organism |
Homo sapiens |
| Characteristics |
tissue: fetal brain
time post-kcl stimulation: 0hr
replicate: 1
Sex: female
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| Treatment protocol |
For KCl depolarization of neurons, neuronal cultures were first silenced overnight in culture media with 1 μM Tetrodotoxin (TTX) and 100 μM D-AP5. The following day samples were incubated for 0, 1 or 6 hours in 55 mM KCl prior to harvest.
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| Growth protocol |
Human neuronal cultures from five and two different individuals were grown for RNA-seq and ChIP-seq experiments, respectively, according to the supplier’s instructions (ScienCell).
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| Extracted molecule |
total RNA |
| Extraction protocol |
The mini-ChIP assays were performed (as described in Adli and Bernstein, 2011) on human neuronal cells that had been cultured for around two weeks. Cells were cross-linked, lysed, sonicated for 3.5 minutes using a Branson 250, and the fragmented chromatin was immunoprecipated overnight with 1ug of H3K27Ac (abcam Cat# ab4728) and H3K27me3 (Millipore Cat# 074490) antibodies. After samples were incubated with Protein A-Sepharose beads for 2 hours they were collected and washed; the DNA was eluted from the beads twice at 65°C for 10 minutes; the eluted chromatin and the "input" samples were reverse cross-linked at 65°C for 5 hours, then Proteinase K digested at 37°C for 2 hours. The ChIP DNA was recovered by standard phenol-chloroform-isoamyl alcohol extraction, then precipitated overnight with ethanol.
The libraries for RNA-Seq were constructed using the PE RNAseq library kit (Illumina).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
For RNA-Seq of human fetal brain cultures, strand-specific paired-end 76-bp reads were mapped to the hg19 genome using BWA (v.0.6.2) allowing up to 5 mismatches; only #1 ends of fragment pairs were used in subsequent analyses. The usual 24 chromosomal alignment targets were supplemented with ~7 million short sequences representing all possible intragenic exonic sets from the hg19 RefSeq annotation whose junctions are spannable by 76-bp reads. Typically 75-95% of all reads in each library were mappable and ~90% of these aligned uniquely; nonuniquely mapped reads were discarded.
The exons for all transcripts assigned to a gene based on the RefSeq annotation for human 37.1 (12 March 2009) were merged (unioned); along with the constructed library of splice-junction sequences these defined each gene's mature-RNA target region. The total number of bases of all reads that overlapped a gene's exonic region were divided by the total length of the region to yield an average read Density (coverage). Normalization of Densities to a standard of 10M 35-bp reads was effected by multiplying the raw Density by (10M/R)*(35/76), where R is the total number of uniquely mapped reads in a sample that did not overlap any RepeatMasker rRNA elements (RPKM units proportional to Normalized Density units via RPKM = Density/0.35).
Note: Some of the human samples derived from distinct sequencing runs using reads of different length. For consistency, reads longer than 76 bp (100 bp and 101 bp) were truncated to 76bp at their 3' ends before .fastq files from different runs were concatenated together.
For RNA-Seq of cultured mouse cortical cells, strand-specific 76-bp paired-end were mapped to the mm9 genome using BWA, with subsequent analyses the same as described for human but based on the RefSeq annotation for mouse 37.1 (5 July 2007).
genome build: hg19
processed data files format and content: Standard bigWig-formatted files (.bw) for both human and mouse RNA-seq samples represent bwa-aligned reads piled up with single-base-pair resolution and then averaged over tiles of width 20bp. Each set of fragment-end (#1 or #2) reads for a sample have their own pair of tracks for POS & NEG strands, i.e., so there are 4 tracks per sample.
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| Submission date |
Feb 25, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Bulent Ataman |
| Organization name |
Harvard Medical School
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| Department |
Neurobiology
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| Lab |
Michael E. Greenberg
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| Street address |
220 Longwood Ave. GB405
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| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
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| Platform ID |
GPL11154 |
| Series (1) |
| GSE78688 |
Activity-dependent transcriptional changes in human neurons |
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| Relations |
| BioSample |
SAMN04516990 |
| SRA |
SRX1601675 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2072621_Bulent_Human_5845F_KCl_0hr_1_hg19_GNM+SPL_r76_span-20_NEG.bw |
60.0 Mb |
(ftp)(http) |
BW |
| GSM2072621_Bulent_Human_5845F_KCl_0hr_1_hg19_GNM+SPL_r76_span-20_POS.bw |
63.4 Mb |
(ftp)(http) |
BW |
| GSM2072621_Bulent_Human_5845F_KCl_0hr_2_hg19_GNM+SPL_r76_span-20_NEG.bw |
62.2 Mb |
(ftp)(http) |
BW |
| GSM2072621_Bulent_Human_5845F_KCl_0hr_2_hg19_GNM+SPL_r76_span-20_POS.bw |
65.5 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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