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Sample GSM3317447

Query DataSets for GSM3317447

Status

Public on Jun 04, 2019

Title

Stage 8 Vegt ChIP-seq replicate 1

Sample type

SRA

Source name

Xenopus tropicalis embryo

Organism

Xenopus tropicalis

Characteristics

developmental stage: Stage 8
tissue: Whole embryo

Treatment protocol

Embryos were cross-linked in 1% formaldehyde in 1/9x MMR at room temperature for 50 minutes with gentle rocking where each microcentrifuge tube contains 200 embryos with 1mL of the formaldehyde solution. Crosslinking reactions were neutralized by the removal of the formaldehyde solution and incubation with 1ml 125mM glycine solution for 10 minutes on ice. Embryos were then washed twice with 400mL of cold RIPA buffer (50 mM Tris-HCl pH7.4, 150mM NaCl, 1mM EDTA, 0.25% sodium deoxycholate, 1% NP40, 0.1% SDS, 0.5 mM DTT, and Roche cOmplete protease inhibitor cocktail). Embryos were flash-frozen in liquid nitrogen and stored at -80°C.

Growth protocol

Xenopus tropicalis females were injected with 10 units of Chorulon (Merck and Co.) 1-3 nights before embryo collection and 100 units of Chorulon on the day of embryo collection. Eggs were collected in a dish coated with 0.1% BSA in 1/9x MMR. The eggs are in vitro fertilized with sperm suspension in 0.1% BSA in 1/9x MMR. The embryos are dejellied with 3% cysteine in 1/9x MMR, pH 7.8, 10 minutes after fertilization and are then ready for manipulation. Embryos were staged using the Nieukwoop-Faber developmental table.

Extracted molecule

genomic DNA

Extraction protocol

The fixed embryos were homogenized in RIPA buffer and incubated on ice for 10 minutes. Samples were then centrifuged at 14,000rpm for 10 minutes at 4°C. Pellets were resuspended in RIPA buffer and sonicated on ice to an average size of 100-500bp. The resulting samples were centrifuged at 14,000 rpm for 30 minutes at 4°C to remove insoluble cellular debris. The sheared chromatin was pre-cleared by incubating with 20 mL Protein G-coated Dynabeads (Invitrogen) for 2 hours at 4°C with rotation. Meanwhile, another 20 mL Protein G Dynabeads were incubated in a tube rotator in 1 mg/mL torula RNA and 0.5% BSA in 1x PBS for 30 minutes at 4°C to block non-specific binding. This second 20 mL Protein G Dynabeads were then pre-bound with 2 mg anti-vegt antibody at 4°C for > 1 hour. The pre-cleared chromatin was added to antibody-bound Dynabeads, and incubated overnight at 4°C on a tube rotator. The next day, the beads were washed, and DNA was eluted off the beads with TE buffer containing 1% SDS, and reverse-crosslinked at 65°C overnight. Sonicated input control was diluted 3-fold with elution buffer and also incubated at 65°C. All samples were treated with RNAse A, Proteinase K, phenol/chloroform extracted, and ethanol precipitated overnight. DNA pellets were resuspended in Qiagen EB solution.
ChIP-seq libraries were generated using Nextflex ChIP-seq kit (Bioo Scientific), analyzed using an Agilent Bioanalyzer 2100, quantified using KAPA qPCR and sequenced using Illumina instruments.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina HiSeq 4000

Description

processed data file: vegt-IDR-optimal-peaks.bed

Data processing

ChIP-seq peak calling and IDR: Reads were aligned to the X. tropicalis genome v9.0 (Hellsete et al., 2010; Xenbase FTP) using Bowtie v2.2.7 (Langmead and Salzberg 2012) with default options and peaks were called against stage 8 input DNA (Charney et al., 2017) using Macs v2.0.10 (Zhang et al., 2008) with the option –p 0.001 but otherwise default options . ENCODE based irreproducibility discovery rate (IDR) was performed using the following p-value thresholds for the following comparisons: 0.01 for original biological replicates, 0.02 for pseudoreplicates of each biological replicate and 0.0025 for pseudoreplicates generated from pooled reads of biological replicates (Li et al., 2011).
Genome_build: Xenopus tropicalis genome v 9.0

Submission date

Aug 02, 2018

Last update date

Jun 04, 2019

Contact name

Kitt D. Paraiso

E-mail(s)

kparaiso@uci.edu

Phone

949-824-7950

Organization name

University of California, Irvine