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Sample GSM2428257

Query DataSets for GSM2428257

Status

Public on Mar 31, 2017

Title

Endoderm_H3K4me3_ChIP_rep2 (Group8)

Sample type

SRA

Source name

Endoderm, H3K4me3 ChIP

Organism

Xenopus laevis

Characteristics

starting material: 100 embryos
developmental stage/condition: stage 18-21 embryo
cell type: endoderm
chip antibody: H3K4me3 (Abcam ab8580, 0.5 ug per 50 embryos)
pairing of samples: Group8

Growth protocol

None

Extracted molecule

genomic DNA

Extraction protocol

ChIP and ChIP-seq preparation: Chromatin Immunoprecipitation (ChIP) was performed as described. Neurula (stage 18) embryos were generated by in vitro fertilization. For each ChIP experiment, 50 embryos were dissected in 1xMBS to obtain the endoderm tissue. Samples were fixed in 2 ml of 1% Formaldehyde in 0.1x MMR for 25 min at room temperature, followed by 4 washes with 1 mL 0.1xMMR and equilibration in 500 μl HEG solution (50mM Hepes-KOH pH 7.5, 1 mM EDTA, 20% Glycerol) at 4°C, then excess buffer was removed and samples were frozen at -80°C. To extract chromatin, the samples were homogenized in 2 ml buffer E1 (50mM Hepes-KOH pH 7.5, 140mM NaCl, 1mM EDTA pH 8.0, 10% Glycerol, 0.5% Igepal CA-630, 0.25% Triton X-100, 1mM DTT, complete protease inhibitors (Roche)). Chromatin was collected by centrifugation for 2 min at 3500 rmp, 4°C and then washed two times with 2 ml E1, three times with 2ml buffer E2 (10mM Tris pH 8.0, 200mM NaCl, 1mM EDTA, 0.5mM EGTA, complete protease inhibitors (Roche)) and three times with 500 μl Buffer E3 (10mM Tris pH 8.0, 200mM NaCl, 1mM EDTA, 0.5mM EGTA, 0.1% Na-deoxycholate, 0.5% N-lauroylsarcosine, complete protease inhibitors (Roche)). Chromatin was fragmented by sonication for 20 cycles (30s on and 30s off) using a Bioruptor (Diagenode). The samples were centrifuged at 15min, 4°C at full speed, the supernatant was collected and Triton X-100 was added to 1%. 25 μl of the solution were put aside to serve as Input for later analysis. Before ChIP, primary anti-H3K4me3 (Abcam ab8580, 0.5 μg per 50 embryos) antibodies were bound to PBS washed magnetic beads conjugated with secondary antibody (Invitrogen 11204D, 25 μl per 50 embryos) in 500 μl 1xPBS 0.1% BSA overnight at 4°C on a rotating wheel. Beads were washed 3 times with 1xPBS 0.1% BSA, added to the fragmented chromatin solution and incubated overnight at 4°C on a rotating wheel. Beads were then washed 6 times with RIPA buffer (50mM Hepes-KOH pH 7.5, 500mM LiCl, 1mM EDTA, 1% Igepal CA-630, 0.7% Na-deoxycholate, complete protease inhibitors (Roche)) and twice with TEN buffer (10mM Tris pH 8.0, 1mM EDTA, 150mM NaCl, complete protease inhibitors (Roche)) for each 10 min. For crosslink reversal, the beads were resuspended in 150 μl Stop buffer (40mM Tris pH 8.0, 10mM EDTA, 1% SDS) and 125 μl Stop buffer was added to the input fraction. The samples were supplemented with Proteinase K (0.3 μg/μl), NaCl (250 mM) and incubated at 65°C overnight. RNase A (DNase free) was added to a final concentration of 200 μg/μl and DNA was Phenol/Chloroform extracted. 150 μg/μl Glycogen was added and DNA was recovered by Ethanol precipitation. The pellet was resuspended in 30 μl H2O, and half was subjected for ChIP-seq library preparation with the TruSeq DNA kit (Illumina, FC-121-2001). Two independent biological replicates were generated for each H3K4me3 ChIP experiment.
Library construction: ChIP-seq library preparation was performed using the TruSeq DNA kit (Illumina, FC-121-2001) according to the manufacturer's protocol.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina HiSeq 1500

Description

Processed data file: IP36_H3K4me3_B_MF_vs_Input12_B_MF_broad_peaks.bed

Data processing

Fastq files processed by filtering out low-quality reads (<Q20) and low-quality bases (<Q20) with sickle. Adaptors were removed with cutadapt 1.0.
RNA-seq data have been aligned to X. laevis genome 6.1 using TopHat 2.0.6.
ChIP-seq data have been aligned to X. laevis genome 6.1 using BWA 0.6.2 with default options. Duplicate reads were then removed.
Peaks were called using MACS2 2.0.9 with --broad options and q-value<0.01.
Differential analysis was conducted using EdgeR package.
The 553,960 assembled transcripts were provided by the International Xenopus Genome Project (http://www.marcottelab.org/index.php/Xenopus_Genome_Project) in October 2012. This assembly was augmented with Xenopus laevis sequences from the NCBI RefSeq database downloaded in February 2012 (30,611 sequences). The combined transcript sequences were filtered with cd-hit-est 4.5.7 (Li and Godzik, 2006) with a similarity score of 95% to remove redundant sequences. This resulted in a final set of 39,384 transcripts. To provide gene names, orthologs were found against the M. musculus proteome (downloaded in January 2013 - NCBI RefSeq) using Inparanoid 4 (Alexeyenko et al. 2006) on predicted ORFs from the Trinity Suite (Grabherr et al. 2011). The sequences were further annotated using InterProScan 4.8 (Zdobnov and Apweiler, 2001) to provide both InterPro Domains (Release 35) and Panther 7.2 ontology terms (Thomas et al. 2003). Xenopus laevis NCBI Descriptions were provided for transcripts that originated at the NCBI. The gtf file containing this information is reported as supplementary material of the previous study: PMID: 27034506, DOI: 10.1101/gr.201541.115.
Genome_build: X. laevis genome 6.1 (ftp://ftp.xenbase.org/pub/Genomics/JGI/Xenla6.1/)
Supplementary_files_format_and_content: *.xls: Excel files contain raw counts.
Supplementary_files_format_and_content: *.bed: BED files contain coordinates of broad peaks called with MACS2 (2.0.9).

Submission date

Dec 13, 2016

Last update date

May 15, 2019

Contact name

Angela Simeone

E-mail(s)

angela.simeone@gmail.com

Organization name

University of Cambridge

Department

Cancer Research UK Cambridge Institute