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Sample GSM1873383 Query DataSets for GSM1873383
Status Public on Feb 25, 2017
Title CAPIP_seq_KO_rep2
Sample type SRA
Source name CAPIP_seq_KO
Organism Mus musculus
Characteristics strain background: 129/Ola
age: E14
genotype/variation: Dnmt3b ko
cell type: Mouse embryonic stem cells (mESCs)
rip antibody: Anti-m3G-cap, m7G-cap (Millipore H-20)
Treatment protocol Generation of Dnmt3b KO ESCs was performed using TALEN technology. In brief, cells were transfected with the two TALEN constructs targeting Exon 17 of murine Dnmt3b and after 16 hours were seeded as a single cell. After 1 week, clones were screened by western blot analyses. Positive clones were analyzed by genomic sequencing of the TALEN target.
Growth protocol Mouse embryonic stem cells E14 were grown on 0.1% gelatin-coated plates and maintained in DMEM (4.5g/L D-Glucose), supplemented with 15% heat-inactivated FBS, 0.1mM NEAA, 1mM Sodium Pyruvate, 0.1mM 2-Mercaptoethanol, 25U/ml penicillin, 25 μg/ml streptomycin and 1,500U/ml LIF
Extracted molecule total RNA
Extraction protocol Total RNA was extracted by using TRIzol reagent (Invitrogen).
For immunoprecipitation of mRNA for CAPIP-Seq experiments, 30ug of total RNA were fragmented by alkaline hydrolysis in ~200nt fragments and incubated with 5ug of mouse anti-CAP antibody (Anti-m3G-cap, m7g-cap, Clone H20, Millipore MABE419) (or IgG) overnight at 4°C in 0,5ml of IP Buffer (10mM Tris-HCl pH 7.5; 150mM NaCl; 0,1% Triton X-100) supplemented with 50U/ml RNaseOUT (Invitrogen), 50U/ml SuperaseIN (Invitrogen), and 50U/ml RNase Inhibitor (Ambion).  25ul of Dynabeads Protein G (Invitrogen) were saturated overnight at 4°C in IP Buffer supplemented with 150ug of Sonicated Salmon Sperm DNA (Qiagen). Following incubation, beads were washed two times in IP Buffer and incubated with the preformed RNA-antibody complexes at 4°C. After 3hrs, beads were washed 4 times with IP Buffer. Specific elution of recovered fragments were obtained by incubation of beads with 100μl Elution Buffer [5mM Tris pH7.5; 1mM EDTA; 0.05% SDS; 0.3mg/ml Proteinase K] for 1h 30’ at 50°C. Fragments were then purified by addition of 1ml TRIzol reagent (Invitrogen), and subjected to random-primed reverse transcription using the SuperScript III Reverse Trancriptase (Invitrogen) at 50°C for 1h. Resulting cDNAs were then used as input for the TruSeq RNA Sample Prep kit (Illumina), starting from the “Second Strand Synthesis” step, to produce the sequencing library, following manufacturer instruction.
Library strategy RIP-Seq
Library source transcriptomic
Library selection other
Instrument model Illumina NextSeq 500
Data processing library strategy: CAP ImmunoPrecipitation (CAPIP)-seq
Basecalls performed using CASAVA version 1.8
Reads were mapped on mm9 using TOPHAT v2.0.6 with the following parameters :--min-anchor=5 --min-isoform-fraction=0.01 --max-multihits=10 --bowtie1
Genome_build: mm9
Supplementary_files_format_and_content: bedGraph files were performed by using Wiggles tool
Submission date Sep 09, 2015
Last update date May 15, 2019
Contact name Francesco Neri
Organization name University of Torino
Street address Via Nizza 52
City Torino
State/province Italy
ZIP/Postal code 10126
Country Italy
Platform ID GPL19057
Series (2)
GSE72852 Intragenic DNA methylation prevents cryptic transcription initiations on gene bodies [CAPIP-seq]
GSE72856 Intragenic DNA methylation prevents cryptic transcription initiations on gene bodies
BioSample SAMN04044195
SRA SRX1213512

Supplementary file Size Download File type/resource
GSM1873383_CAPIP_seq_KO_rep2.bedGraph.gz 12.8 Mb (ftp)(http) BEDGRAPH
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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