GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM2029147

Query DataSets for GSM2029147

Status

Public on Jan 07, 2016

Title

H3K4me3-poly_rep2-B

Sample type

SRA

Source name

HeLa cells

Organism

Homo sapiens

Characteristics

cell line: HeLa cells
treatment: Paraformaldehyde fixation (1%, 30 minutes)
chip antibody: H3K4me3 polyclonal Ref: Active motif 39159

Treatment protocol

40 million cells were sonicated in 500μL of Lysis Buffer (1% Na-deoxychlorate, 50mM TrisHCl pH8, 140mM NaCl, 1mM EDTA, 1% Triton X-100) containing 5-times diluted Protease Inhibitor Cocktail (PIC; Roche Diagnostic; 1 tablet solubilized in 10ml Lysis Buffer). Sonication was performed with a Bioblock Scientific instrument (Vibra Cell 75043; 40 cycles, 30s ON end 59s OFF; 38% power). Chromatin fragmentation was evaluated by agarose gel electrophoresis as follows: 20μL of sonicated chromatin was diluted with 20μL TE (10mM Tris-HCl pH 8, 1mM EDTA) and 5μL 5M NaCl was added. Diluted chromatin was incubated at 100°C for 30 min, centrifuged at 12,000 rpm at room temperature and the supernatant was loaded onto a 2% agarose gel.

Growth protocol

HeLa cells were grown in DMEM 1g/L glucose, 5% Fetal Calf Serum and 40μg Gentamicin to a density of 15–20 millions cells/15cm plates. Cells were fixed for 30min with paraformaldehyde (1% in PBS). Fixation was quenched with 0.2M glycine in PBS, then cells were washed three times with PBS, collected and stored at -80°C.

Extracted molecule

genomic DNA

Extraction protocol

25μL of ChIP-IT Protein G Magnetic Beads (ActiveMotif) were incubated with the antibody under evaluation (the amounts of antibody in use corresponded to that indicated by the supplier’s information) in presence of 100μL PIC-containing Lysis Buffer. After two hours at 4°C on a rotating shaker, chromatin from 3 million cells was added and the final vol- ume was adjusted to 500μL with PIC-containing Lysis Buffer and incubation on a rotating shaker was continued overnight at 4°C. The immunoprecipitated chromatin was recovered by magnetic bead separation, followed by multiple washing steps on a custom liquid handling platform (TECAN EVO75). Specifically, the washing is performed as follows: (1) Low salt washing (0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM TrisHCl pH 8, 150mM NaCl); (2) High salt washing (0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM TrisHCl pH 8, 500mM NaCl); (3) LiCl-washing (0.25M LiCl, 1% IGEPAL CA630, 1% Na-deoxycholate, 1mM EDTA, 10mM Tris pH 8) and (4) 1×TE washing. The immunoprecipitated chromatin was eluted and de-crosslinked in 100μL of elution buffer (1% SDS, 100mM NaHCO3, 250mM NaCl, 0.2mg/ml Proteinase K) and incubated for 4 hours at 65°C. The eluted chromatin was supplemented with 200μL H2O and 300μL phenol/chloroform/isoamyl alcohol (25/24/1) mix was added. After two extraction steps, the aqueous phase was subjected to ethanol precipitation in presence of 1μL GlycoBlue (Invitrogen; 15mg/ml). The precipitated material was re-suspended in 45μL H2O; 5μL was used for validation by quantitative PCR, the remaining 40μL was used for DNA library preparation.
The DNA library preparation for massive parallel sequencing was performed according to standard procedures (NEXTFlex ChIP-Seq Kit (Biooscientific)) adapted to automation by our custom liquid handling platform (TECAN EVO75). Prior to DNA sequencing library preparation was monitored using a Tapestation (Agilent). Samples were sequenced on an Illumina HiSeq 2000 platform following manufacturer’s standard procedures and certified on the basis of the NGS-QC Generator concept (Mendoza-Parra et al; NAR 2013).

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina HiSeq 2000

Description

H3K4me3-poly_rep2.bw

Data processing

ChIP-seq and related datasets (IP and Input control) were quality certified with NGS-QC Generator tool (www.ngs-qc.org). Specifically, this methodology allows providing enrichment quality descriptors discretized in a scale ranging from “AAA” (Best) to “DDD” (worst). The quality scores reports associated to these datasets are available at : http://www.ngs-qc.org/certdb.php
bigWig files were generated with a read elongation of 150nts and a resolution of 20nts
Genome_build: hg19
Supplementary_files_format_and_content: bigWig files

Submission date

Jan 07, 2016

Last update date

May 15, 2019

Contact name

Marco Antonio Mendoza-Parra

E-mail(s)

marco@igbmc.fr

Organization name

IGBMC

Street address

1, rue Laurent Fries