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| Status |
Public on Apr 15, 2010 |
| Title |
KCl_hr6_total_RNA_b3 |
| Sample type |
SRA |
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| Source name |
7 DIV E16.5 mouse cortical neurons, before and after membrane depolarization induced by extracellular potassium
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| Organism |
Mus musculus |
| Characteristics |
protein immunoprecipitated: NA
antibody: NA
strain: C57B6
agent: untreated
developmental stage: DIV 7, E16.5
cell type: cortical neuron
biological rep: 3
technical rep: 1
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| Treatment protocol |
0, 1, 2, or 6 hours of 55mM KCl, with overnight quieting with TTX/APV
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| Growth protocol |
7 DIV E16.5 mouse cortical neurons grown in B27/NB on PO-coated plastic 15-cm plates
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| Extracted molecule |
total RNA |
| Extraction protocol |
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, 5–10ug of RNA isolated from mouse cortical 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 with 10–18 minutes 37° RNAseIII treatment, and 10 minutes of RNAseIII inactivation at 65°. 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. Mouse cortical neuron WT-Seq data presented in this manuscript are from one specific biological replicate, but each result was confirmed in at least one additional replicate.
Chromatin immunoprecipitation-sequencing (ChIP-Seq): Forty million mouse cortical neurons cultured to in vitro day 7 were used for each ChIP-Seq library construction. ChIP was performed as described2 using antibodies listed above. The immuno-precipitated DNA fragments were repaired by the End-It DNA End Repair Kit (Epicentre Biotechnology) according to the manufacturer’s instructions. The end-repaired ChIP DNA fragments were purified by MinElute Reaction Cleanup Kit (Qiagen) and eluted in 20 ul in EB buffer. The resulting DNA fragments were ligated with P1 and P2 adaptors for SOLiD genome analyzer (adaptor sequences can be available upon request) for 20 minutes at room temperature using the Quick Ligase Kit (NEB), followed by purification using MinElute Reaction Cleanup Kit (Qiagen). The purified, adaptor-ligated ChIP DNA fragments were subject to 6% native-PAGE for an in-gel PCR reaction. A gel slice containing 175–200 bp adaptor-ligated ChIP DNA fragments (corresponding to 125–150bp genomic fragment sizes) was cut and shredded. PCR Platinum Supermix (100–200 ul, Invitrogen), 50 pmoles of PCR primers (available upon request), 0.5 ul Taq DNA polymerase (NEB), and 0.15 ul Pfu Turbo DNA polymerase (Stratagene) were added into the shredded gel slice. The adaptor-ligated ChIP DNA fragments were amplified by 15 cycles of in-Gel PCR. After the PCR reaction, gel pieces were filtered out by 0.45 um filter spin column, and the amplified ChIP-Seq library was purified by MinElute PCR purification kit (Qiagen). The library was purified by one more round of 6% PAGE. A gel slice containing 200–250 bp PCR products (110–150bp fragment size) was cut and shredded, and the amplified library was extract out of the gel by passive elution in elution buffer (1.5M ammonium acetate in 1X TE). Gel pieces were filtered out by filter spin column, and the resulting ChIP-Seq library was purified using the Qiaquick PCR purification kit (Qiagen).
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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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| Description |
total RNA
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| Data processing |
Corona lite read alignment to mm9; discard non-uniquely aligned reads; conversion of uniquely aligned reads (0-3 mismatches) into bigWig (.bw) file
For ChIP-Seq, the .bw file reflects extended 120bp fragments corresponding to each ChIP-Seq read
For RNA-Seq, the .bw files contain information only about the 5' most base of each read; there are separate bigWig files for + and - genomic strands
The .bedgraph files from which the .bw files were derived are also provided.
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| Submission date |
Apr 02, 2010 |
| Last update date |
May 15, 2019 |
| Contact name |
jesse M gray |
| E-mail(s) |
jesse.gray@gmail.com
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| Phone |
6174321877
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| Organization name |
Harvard Medical School
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| Department |
Genetics
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| Lab |
Gray lab
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| Street address |
77 avenue louis pasteur, NRB 356
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| City |
boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
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| Platform ID |
GPL9318 |
| Series (1) |
| GSE21161 |
Widespread transcription at neuronal activity-regulated enhancers |
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| Relations |
| SRA |
SRX019182 |
| BioSample |
SAMN00011268 |
| Named Annotation |
GSM530213_hr6_b3+.bw |
| Named Annotation |
GSM530213_hr6_b3-.bw |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM530213_hr6_b3+.bedgraph.gz |
56.5 Mb |
(ftp)(http) |
BEDGRAPH |
| GSM530213_hr6_b3+.bw |
235.1 Mb |
(ftp)(http) |
BW |
| GSM530213_hr6_b3-.bedgraph.gz |
57.7 Mb |
(ftp)(http) |
BEDGRAPH |
| GSM530213_hr6_b3-.bw |
235.5 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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