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| Status |
Public on Jan 01, 2016 |
| Title |
V6.5_Aff3_shRNA-rep2 (nascent RNA) |
| Sample type |
SRA |
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| Source name |
ES cell culture
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| Organism |
Mus musculus |
| Characteristics |
strain: v6.5
cell type: mouse embryonic stem cell
shRNA: Aff3
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| Treatment protocol |
none
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| Growth protocol |
The mouse ES cell lines used in the current study were grown on irradiated primary mouse embryonic fibroblast (MEF) feeder layers in 0.1% gelatin-coated tissue culture vessel. Mouse ES cells were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 15% ES-certified fetal bovine serum (Hyclone), recombinant LIF (Millipore), 2 mM L-glutamine, 0.1 mM nonessential amino acids and 0.1 mM β-mercaptoethanol. For experimental analyses, ES cells were grown for one passage without MEF feeder cells in tissue culture vessel for 30 minutes. The uniparental MEF cell lines were cultured in DMEM supplemented with 10% FBS (Sigma).
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| Extracted molecule |
total RNA |
| Extraction protocol |
For ChIP-Seq, 5×107 cells were used per immunoprecipitation according to previously described protocols (Lee et al., 2006). ChIP-Seq libraries were prepared with Illumina's kit or KAPA kit. For RNA-seq, cells were infected with lentivirus carrying either Non-targeting shRNA, Aff3 shRNA in the presence of 8 ug/ml of polybrene. 24 hours later, cells were selected with 2 ug/ml of puromycin for additional 48 hours and then ES were grown one passage off feeders for 30 min before harvesting. Total RNA was isolated with the RNeasy (74106 Qiagen) kit, treated with DNase I (M0303L NEB), and re-purified with RNeasy. For total RNA-seq analyses, total RNA was depleted of ribosomal RNA using Ribozero (Illumina) before library preparation performing Tru-seq sample prep (Illumina). For Nascent RNA-seq, about 1×108 fresh ES cells were washed twice in ice-cold PBS. The cells were then resuspended in Buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.5 mM DTT and proteinase inhibitors (Roche)) and incubated on ice for 15 min. Cell suspension was transferred to a pre-cold Dounce tissue grinder and homogenized 10 times by using a tight pestle on ice. After centrifugation the pellet containing enriched nuclei was washed twice in S1 buffer (0.25 M Sucrose, 10 mM MgCl2, 10 mM HEPES, pH 7.9, 1 mM DTT and proteinase inhibitors). To obtain the chromatin pellet, the nuclei pellet was then washed in ice-cold NUN buffer (20mM HEPES pH7.9, 7.5 mM MgCl2, 0.2 mM EDTA, 300 mM NaCl, 1 M Urea, 1% NP-40, 1 mM DTT, 20U/ml RNase Inhibitor (Ambion), and proteinase inhibitors) for 5 times. Chromatin bound RNA was isolated with RNeasy kit. The RNA was poly-A depleted by using Oligo(dT) magnetic beads (Invitrogen) and treated RNase free DNase I, and re-purified with the RNeasy column. 500 ng RNA was used for ribosomal RNA depletion with Ribo-zero Gold (Epicentre) and library preparation was performed with TruSeq Stranded Total RNA-seq Sample prep kit.
ChIP-seq, total RNA-seq and nascent RNA-seq libraries were prepared for sequencing using standard Illumina or KAPA protocols
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
Nascent RNAseq after Aff3-RNAi
Ribosomal RNA-depleted nascent RNA
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| Data processing |
Base-calling and quality filtering, default settings, Illumina Casava 1.8
ChIP-seq reads were aligned using Bowtie v0.12.9, allowing unique reads only and up to three mismatches of a 50bp seed length.
RNA-seq and Nascent RNA-seq alignments were done using TopHat v2.0.9 with bowtie1 v0.12.9, using -g 1 option and allowing up to two mismatches.
[ChIP-seq] Alignments were extended to a total fragment length of 150 bases in the orientation of the read alignment. UCSC bigWig files were created at 1bp resolution and normalized to total alignable reads (reads-per-million).
[nascent-Seq and RNA-seq] Reads were aligned using Ensembl 67 GTF mouse annotations using TopHat v2.0.9 and Bowtie v0.12.9. Alignments were not extended. UCSC bigWig files were created at 1bp resolution and normalized to total alignable reads (reads-per-million).
Genome_build: UCSC mm9
Supplementary_files_format_and_content: http://genome.ucsc.edu/goldenPath/help/bigWig.html
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| Submission date |
Dec 23, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Joan Conaway |
| E-mail(s) |
JLC@stowers.org
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| Organization name |
Stowers Institute
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| Street address |
1000 E 50th ST
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| City |
Kansas City |
| State/province |
MO |
| ZIP/Postal code |
64110 |
| Country |
USA |
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| Platform ID |
GPL13112 |
| Series (1) |
| GSE64489 |
Allele-specific regulation of gene expression through enhancer function and transcriptional elongation control at imprinted loci |
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| Relations |
| BioSample |
SAMN03272954 |
| SRA |
SRX823763 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1572321_V6.5_Aff3_shRNA-rep2-negativeStrand.bw |
24.3 Mb |
(ftp)(http) |
BW |
| GSM1572321_V6.5_Aff3_shRNA-rep2-positiveStrand.bw |
25.2 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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