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Sample GSM1838884

Query DataSets for GSM1838884

Status

Public on Jul 31, 2015

Title

KO E17.5 brain SMC1-R2_ChIP-seq

Sample type

SRA

Source name

whole brain

Organism

Mus musculus

Characteristics

tissue: whole brain
strain: C57BL/6
age: E17.5 embryo

Extracted molecule

genomic DNA

Extraction protocol

ChIP was performed in tissues (cerebral cortex and pancreas, in the case of 8-10 week old C57Bl/6 mice, and whole brain in the case of E17.5 embryos) from wildtype, SA1 heterozygous and SA1-null mice with custom made rabbit polyclonal antibodies against SA1 and SMC1 and RNApolIISer2P (AB5095, Abcam) as described (Cuadrado et al. 2010). Mice were housed in a pathogen-free animal facility following the animal care standards of the institution. Fresh tissues collected in 6 MW plates containing cold PBS with 1 mM PMSF and protease inhibitor cocktail (Roche) were immediately minced to get small fragments of about 1 mm diameter. Tissue pieces (pooled from three individuals) were centrifuged at 2000 rpm for 2 minutes at 4ºC, resuspended in 1 ml of fixing solution (1% formaldehyde, 50 mM HEPES-KOH, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA) and incubated 20 min at room temperature in a rotating wheel. Cross-linking was stopped by adding 1/20 volume of 2.5M Glycine for 5 minutes at RT. Tissue pieces were washed twice with cold PBS, recovered by centrifugation at 3000 rpm for 7 minutes at 4ºC, resuspended in 2 ml lysis buffer (1% SDS, 10mM EDTA, 50 mM Tris-HCl pH 8.1; 2x107 cells per ml) and sonicated in Covaris system (shearing time 30 min, 20% duty cycle, intensity 10, 200 cycles per burst and 30 seconds per cycle).
DNA samples were provided by the user. 15 ng of ChIP DNA as quantitated by fluorometry was used. Electrophoretic fraction of 100-200bp was processed through subsequent enzymatic treatments of end-repair, dA-tailing, and ligation to adapters as in Illumina's "TruSeq DNA Sample Preparation Guide" (part # 15005180 Rev. C). Adapter-ligated library was completed by limited-cycle PCR with Illumina PE primers (11 cycles). The resulting purified DNA library was applied to an Illumina flow cell for cluster generation (TruSeq cluster generation kit v5) and sequenced on the Genome Analyzer IIx with SBS TruSeq v5 reagents by following manufacturer's protocols.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina Genome Analyzer IIx

Description

Processed data file: KO_SMC1_Losada.bed
Chromatin immunoprecipitation

Data processing

Read files were quality-checked with FastQC.
Reads where aligned to mm9 with Illumina's Eland
Same sample replicas where merged
Peaks were called with MACS 1.4 - All samples where compared to Input - Only peaks with FDR <=5% were retained
Genome_build: GRCm37/mm9
Supplementary_files_format_and_content: BED files with mm9 coordinates of peaks with FDR <= 5%

Submission date

Jul 30, 2015

Last update date

May 15, 2019

Contact name

Ana Losada

E-mail(s)

alosada@cnio.es

Phone

+34 - 917 328 000

Organization name

Centro Nacional de Investigaciones Oncológicas (CNIO)