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| Status |
Public on Apr 20, 2015 |
| Title |
WT-RA_DNA |
| Sample type |
SRA |
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| Source name |
46C mES cells
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| Organism |
Mus musculus |
| Characteristics |
sample type: embryonic stem cells (RA, day1)
treatment: LIF withdrawal, 2uM RA, day1
strain: 129/Ola
chirp probes: ChIRP probes targeting Haunt mRNA
genotype: wild type
cell line: 46C
cell type: embryonic stem cells
chip antibody: none
barcode: TCACGTT
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| Treatment protocol |
Cells were washed once with cold PBS, then harvested by trypsin digestion.
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| Growth protocol |
Wild-type ESCs or Haunt KO ESCs were maintained in strandard ES medium supplemented with 15% heat-inactivated FCS, 1% of nucleoside mix (100 × stock, Millipore), Penicillin-Streptomycin Solution (100 × stock, Life Technologies), 2 mM Glutamax (100 × Life Technology), 0.1 mM non-essential amino acid, 0.1 mM 2-mercaptoethanol and supplied with 1000 U/ml recombinant leukemia inhibitory factor (LIF, millipore).
Wild-type ESCs or Haunt KO ESCs were maintained in strandard ES medium supplemented with 15% heat-inactivated FCS, 1% of nucleoside mix (100 × stock, Millipore), Penicillin-Streptomycin Solution (100 × stock, Life Technologies), 2 mM Glutamax (100 × Life Technology), 0.1 mM non-essential amino acid, 0.1 mM 2-mercaptoethanol and supplied with 1000 U/ml recombinant leukemia inhibitory factor (LIF, millipore).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIRP was done as described previously with the following modifications (Chu et al. 2011). First, 59 nt DNA probes were biotinylated through terminal transferase (New England Biolabs). Intensive crosslinking of cells was performed in the following sequence: two rounds of 3000J UV treatment on ice, 0.8% of formaldehyde (FMA) for 10 minutes, 2mM of DSP (Dithiobis [succinimidyl propionate]) for 30 minutes, and followed by 3.7% of FMA for 10 minutes at room temperature. Crosslinked cell pellets were lysed. Chromatin was fragmented into a range of 2 - 5 kb by sonication. Hybridization, washing and elution steps were performed as described previously, except that we included an additional stringent wash (0.1x SSC). After elution and reverse crosslinking, RNA was further extracted by TRIzol reagent (Life Technologies).
The library was constructed by library preparation modules (New England Biolabs) according to manufacturer's instructions. Briefly, ChIRP retrived RNA was fragmentated by NEBNext Magnesium RNA Fragmentation Module at 94 ℃ for 210 seconds. The fragementated RNA was reverse transcribed by SuperScript III First-Strand Synthesis System for RT-PCR kit (Life Technology), 2'nd strand was then synthesized by NEBNext mRNA Second Strand Synthesis Module , the end repair, dA-tailing and adaptor ligation were also following the instruments by respect module. the adaptor for the libary was sythersized from IDT with the first 7nt is "TCACGTT" as a barcode. After liagation, the DNA was amplified by primer 1.0 (AATGATACGGCGACCACCGAGATCTACAC, synthersized from IDT) and 2.0 (CAAGCAGAAGACGGCATACGAGAT, synthersized from IDT) for 15 cycles. After this, the product was further purified and size selected by Ampure XP beads (Beckman Coulter). The library was sequenced on the Genome Analyzer following the manufacturer's protocols.
Total RNA of RNA-Seq were extracted by TRIzol reagent according to manufacturer's instruction. mRNA were further isolated by Dynabeads mRNA purification kit (Life Technologies) according to manufacturer's instruction.
Libraries were prepared by illumina TruSeq Stranded mRNA LT Sample Prep Kit (RS-122-2101) according to the standard protocols of the manufacturer. ChIRP retrieved DNA-seq were prepared by library preparation modules (New England Biolabs) according to manufacturer's instructions.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Basecalls performed using CASAVA version
ChIRP retrived DNA-Seq reads were aligned to the mm9 genome assembly using Bowtie version 1.0.0 with the default parameters
RNA-seq reads were aligned to the mm9 genome assembly using Tophat v2.0.11,allowing for uniquely mapped reads and mapping both spliced and unspliced reads.
FPKM was calculated by Cufflink 2.1.1
Peaks of each sample were called using MACS v. 1.4.2
Genome_build: mm9
Supplementary_files_format_and_content: tab-delimited text files include RPKM values for wild-type or Haunt KO Samples, bedgraph files were generated using MACS v. 1.4.2, including peaks called by default parameters.
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| Submission date |
Feb 09, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
jinlong lu |
| E-mail(s) |
bioyuyang@hotmail.com
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| Phone |
5853098469
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| Organization name |
University of Rochester
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| Lab |
Gorbunova & Seluanov Labs
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| Street address |
213 Hutchinson Hall
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| City |
Rochester |
| State/province |
NY |
| ZIP/Postal code |
14627 |
| Country |
USA |
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| Platform ID |
GPL17021 |
| Series (1) |
| GSE58514 |
Opposing roles for the lncRNA Haunt and its genomic locus in regulating HOXA gene activation during embryonic stem cell differentiation |
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| Relations |
| BioSample |
SAMN03333453 |
| SRA |
SRX869303 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1604256_Haunt_ChIRP_RA.bedgraph.gz |
37.1 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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