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Sample GSM1180933

Query DataSets for GSM1180933

Status

Public on Sep 19, 2013

Title

INPUT_XBRA12.1

Sample type

SRA

Source name

Input for Xbra ChIP-Seq (gastrula)

Organism

Xenopus tropicalis

Characteristics

strain: Nigerian (Cam4)
genotype/variation: wild type
tissue: embryo
developmental stage: gastrula (st. 11-12.5)
chip antibody: none

Treatment protocol

Embryos were washed in 0.01x MMR and fixed at room temperature with fresh formaldehyde (molecular biology grade; Sigma F-8775) at a final concentration of 1% (v/v). The fixation time was determined empirically according to the developmental stage of the embryo to achieve efficient chromatin shearing and ChIP enrichment. Early developmental stages were fixed over a longer time period than later stages: X. tropicalis gastrula (stage 11-12.5) for 20-25 min and early tailbud (stage 19-20) for 15 min. Fixative solution was aspirated and embryos quickly washed 3-4 times with cold 0.01x MMR. Batches of approximately 250 embryos were equilibrated in ice-cold HEG solution (50 mM HEPES-KOH pH7.5, 1 mM EDTA pH8.0, 20% glycerol). Once the embryos settled, excess HEG was removed. Embryo batches were snap-frozen in liquid nitrogen and stored at -80C for future use.

Growth protocol

Approximately 3000 dejellied embryos were grown for each ChIP-Seq experiment to the indicated developmental stage in 0.05x MMR at 22C.

Extracted molecule

genomic DNA

Extraction protocol

Cross-linked chromatin was extracted with E1, E2 and E3 (Lee et al., 2006 , PMID:17406303). Chromatin was solubilised and sheared to fragments of 100-500bp peaking around 200 bp. ChIP was performed with ChIP-grade antibodies coupled to Dynabeads M-280. Beads were extensively washed with 10 times pre-chilled RIPA buffer (50 mM Hepes-KOH pH7.5, 500 mM LiCl, 1mM EDTA, 1% Igepal CA-630, 0.7% (w/v) Na-deoxycholate) and twice in pre-chilled TEN (10 mM Tris pH8.0, 1 mM EDTA, 150 NaCl). Bound chromatin was eluted from the beads with elution buffer (50 mM Tris pH8.0, 1 mM EDTA pH8.0, 1% SDS) at 65C for 30 min. ChIP and input samples were supplemented with 1/20 volume of 5 M NaCl before incubating them for 6-15 h at 65C to reverse cross-links. One volume of TE pH 8.0 and 200 µg/ml of CONCERT RNase A (Invitrogen) was added to the samples for a 1-h incubation at 37C. The samples were further treated with 200 µg/ml of proteinase K (Ambion) for 2-4 h at 55C. Eventually, the DNA fragments were purified with phenol:chloroform:isoamylalcohol and precipitated in ethanol for sequencing. The air-dried DNA pellet was dissolved in 32 µl of molecular-grade water. The amount of DNA was quantified using a Qubit fluorometer (Invitrogen) following the manufacturer’s instructions. The yield of co-immunoprecipitated DNA was about 10 ng, which created robust libraries.
The single-end library was generated based on the manufacturer’s instructions (Illumina) with minor modifications. DNA fragments were end-repaired, ligated to adaptors at a final dilution of 1:480, size-selected to 200-300 bp by E-gel (Invitrogen) and PCR (18 cycles) amplified. The DNA Clean & Concentrator-5 kit (Zymo Research) was used to clean up reactions. DNA was eluted from the column at 50C. Upon solid phase reversible immobilisation (SPRI) purification, the integrity of the library was checked on the Agilent 2100 Bioanalyzer using an Agilent DNA 1000 chip.
ChIP-Seq libraries were sequenced for 40 cycles on the Illumina Genome Analyser IIx.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina Genome Analyzer IIx

Description

Input for Xbra ChIP-Seq (gastrula)

Data processing

Basecalls were performed using Casava version 1.8.
ChIP-seq reads were aligned to the X. tropicalis genome assembly 4.1 using CLC Bio Genomics Workbench 5.5 default settings. Non-specific and ambiguous matches were ignored.
ChIP-Seq peaks were identified using MACS 2.0.4 (Zhang et al., 2008, PMID:18798982). Settings were as follows: effective genome size, 1,700,000,000bp; band width, 300bp; Model fold, 3-30; Range for calculating regional lambda, 1,000-10,000bp; FDR, 0.01.
Genome_build: X. tropicalis JGI v4.1 (August 2005, JGI4.1/xenTro2, available on the UCSC Genomic Browser)
Supplementary_files_format_and_content: BigWig files with -log(peak p-value)

Submission date

Jul 05, 2013

Last update date

May 15, 2019

Contact name

George E. Gentsch

E-mail(s)

[email protected]

Organization name

The Francis Crick Institute

Department

Developmental Biology Laboratory