GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM3934952

Query DataSets for GSM3934952

Status

Public on Sep 04, 2020

Title

YueLab-Input-Brain-rep1

Sample type

SRA

Source name

Tissue

Organism

Danio rerio

Characteristics

strain: Tuebingen
tissue: Brain

Growth protocol

Ault Tuebingen zebrafish were raised under standard laboratory conditions

Extracted molecule

genomic DNA

Extraction protocol

All the tissues were ground in the liquid nitrogen and fixed by 1% formaldehyde at room temperature for 15 min. 0.2 M glycine was added and incubated at room temperature for 5 min to quench the fixation. Fixed tissues were then washed once by cold 1X PBS. Tissue pellet was resuspended and incubated on ice for 10 min in 100 µL of ChIP-seq lysis buffer (20 mM Tris-HCl, pH 8.0, 1% SDS, 50 mM EDTA, 1X proteinase inhibitor cocktail). Next, 900 µL cold 1X TE buffer was added to dilute SDS concentration and the nuclei suspension was sonicated using Covaris E220 with the following parameters: 140 W, duty factor 5, 200 per burst. Sonication time is variable, depending on tissue types. To check the chromatin fragmentation size, 20 µL of input chromatin was reverse crosslinked in elution buffer (20 mM Tris-HCl, pH 8.0, 1% SDS, 1 mM EDTA) at 65 °C overnight, treated with Rnase A and proteinase K and purified by phenol-chloroform extraction. Input DNA was then loaded on a Lonza flash gel to make sure that the majority of DNA size distributes between 100-300 bp. To prepare antibody-beads complex, 3 µg of histone H3K27ac antibody (Active Motif, 39133) or H3K4me3 antibody (EMD Millipore, 07-473) was mix with 12 µL M-280 sheep anti-rabbit IgG Dynabeads (ThermoFisher, 11203D) in 150 µL of 5 mg/mL BSA/1X PBS buffer, with rotation at 4 °C for 3 h. After incubation, antibody-beads complex was washed once with BSA/1X PBS buffer. About 200 µg chromatin was used per IP. Equal volume of master mix (1X TE, 2% Triton X-100, 0.2% sodium deoxycholate, 2X proteinase inhibitor cocktail) was mixed with 200 µg chromatin and then incubated with antibody-beads complex overnight with rotation. The next morning, the beads were washed 5 times with cold RIPA wash buffer (20 mM Tris-HCl, pH 8.0, 1% NP-40, 0.7% sodium deoxycholate, 500 mM LiCl, 1 mM EDTA, 1X proteinase inhibitor cocktail). Then the chromatin bound on the beads was eluted by 150 µL of elution buffer at 65 °C for 30 min.
To prepare the library, eluted chromatin was reverse crosslinked and purified by phenol-chloroform extraction. Then DNA was end-repaired by END-IT DNA end-repair kit (Epicentre, ER81050) according to kit’s protocol, added A using Klenow fragment (3’->5’ exo-) (NEB, M0212S), ligated with Illumina TruSeq adaptor (Illumina, FC-121-3001) and subsequently amplified by PCR (Roche, kk2601). The quality and quantity of all the libraries were checked using BioAnalyzer High Sensitivity DNA Kit (Agilent). The libraries were sequenced on Illumina HiSeq 2500 with reads length of 2X 50 bp or 2X 100 bp.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina Genome Analyzer

Data processing

RNA-seq reads were aligned to zv10 genome assembly using STAR;
ChIP-seq and ATAC-seq reads were aligned to zv10 genome assembly using BWA
HiC reads were aligned to zv10 genome assembly using Bowtie2
The TPM value of gene expression was caculated using RSEM
ChIP-seq and ATAC-seq peaks were called using MACS2 with the following setting: ChIP-seq q-value <10e-2, p-value<10e-5, Change>1,FC>2. ATAC-seq: q-value<10e-2 and p-value<10e-5
HiC matrix was generated using HiC-Pro
Genome_build: zv10
Supplementary_files_format_and_content: tab-delimited text files include TPM values for each Sample; the narrowPeak files included the peaks for each Sample; The .hic file were the matrix of Hi-C for each Sample.**All replicates were merged

Submission date

Jul 09, 2019

Last update date

Sep 04, 2020

Contact name

Yu Luan

E-mail(s)

yu.luan@northwestern.edu

Organization name

Northwestern University Feinberg School of Medicine

Department

Department of Biochemistry and Molecular Genetics