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Sample GSM1592487

Query DataSets for GSM1592487

Status

Public on Aug 18, 2016

Title

15_Pol2_EGF20_repl_1

Sample type

SRA

Source name

HeLa S3_Pol2_EGF20'

Organism

Homo sapiens

Characteristics

cell line/vendor: HeLa S3 (ATCC CCL2.2)
stimulated with: EGF (100 ng/ml) for 20min
chip antibody: Pol 2 (4H8)
chip antibody vendor: Santa cruz
chip antibody cat. #: sc-47701

Treatment protocol

Cells were starved for 48 hours in serum-free medium and then stimulated for 20 minutes with EGF (100 ng/ml) (Sigma; E9644).

Growth protocol

Hela S3 cell were grown in DMEM F-12 Ham media (Sigma; D8437) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and penicillin-streptomycin at 50 IU/ml in a humidified 5% CO2 atmosphere at 37oC.

Extracted molecule

genomic DNA

Extraction protocol

1E+07 cells were crosslinked with 1% formaldehyde for 10 minutes at then formaldehyde quenched with 125mM glycine for 5 minutes room temperature. After harvest cells were spun down at 1600 x g then washed with 5 ml PBS, spun down again and stored at -80oC as pellets. The pellet was suspended in a 2 mL of hypotonic buffer A [10 mM HEPES, pH 7.9, 2 mM MgCl2, 2 mM KCl and NP-40 0.5% vol/vol] supplemented with protease and phosphatase inhibitors (Thermo; 78441) and suspension was kept for 5min on ice then spun down at 10 000 x g / 4 oC for 3 min. Pellets of nuclei were resuspended in lysis buffer [12.5 mM Tris-HCl, pH 8.0, 2.5 mM EDTA, and 0.1% SDS vol/vol] containing protease and phosphatase inhibitors (Thermo, 78441). Chromatin was sheared in a Bioruptor Plus (Diagenode) using a 30 sec on-off cycle for 15 min at high intensity then centrifuged 12 000 x g for 15 min at 4 °C, aliquoted and stored at -80oC. A modified Fast-ChIP protocol was used for the ChIP-Seq libraries . Briefly, a volume of chromatin (20-30 µl) with equivalent of 5E+06 cells was adjusted to 200 µl with IP buffer [150 mM NaCl, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, NP-40 (0.5% vol/vol), Triton X-100 (1.0% vol/vol), (pH 7.6)] containing protease and phosphatase inhibitors (Thermo) and incubated with 5µg of a given antibody for 30 minutes in ultrasonic bath (Branson, B3510) at 4 oC. Next, the chromatin was cleared by centrifugation at 12 000 x g for 10 min at 4 °C and transferred to a new tube containing 20µl of Dynabeads® Protein A (Life Technologies). Tubes were then rotated at 4 °C for 60 min on a rotating platform (15 rotations per min). Beads were separated on a magnetic stand and washed five times with 800 µl of ice cold IP buffer. DNA was recovered by beads incubation with 50 µl elution buffer [0.5% SDS, 0.1M NaHCO3, 20mg/ml Proteinase K] at 56oC for 30 min with mixing (1000rpm) in a ThermoMixer (Eppendorf). Sample was separated on a magnetic stand, transferred to a PCR tube and then NaCl was added to final concentration of 0.2M followed by overnight reverse crosslinking at 65oC in a thermocycler. DNA was cleaned up with ChIP DNA Purification Kit (Active motif; 58002) according to manufacturer's protocol and DNA concentration was determined with Qubit dsDNA HS Assay Kit (Life Technologies) while the respective size distribution with High Sensitivity DNA Analysis Kit on Bioanalyzer 2100 (Agilent). The antibodies used in ChIP studies were as follow: non-specific rabbit IgG (I-1000, Vector Labs), Pol 2 (4H8) (Santa cruz; sc-47701), pEGFR (Tyr845) (Cell signaling; D63B4), pBRAF (Thr 598/Ser 601) (Santa cruz; sc28006-R), pMek1/2 (Ser217/221) (Cell signaling; 9121S), pErk1/2 (Thr202/Tyr204) (Cell signaling; 4370P).
DNA libraries were prepared from up to 20 ng of DNA using TruSeqTM ChIP Sample Prep Kit (Illumina Inc.), according to standard protocol with modifications. Briefly, DNA fragments were end repaired into blunt ends and single ‘A’ nucleotide was added to the 3’ ends. Adenylated fragments were ligated with Illumina indexed adapters and then amplified during 18-cycles PCR for selective enrichment. Finally, library fragments with insert length of about 120-200 bp were automatically selected using LabChip XT DNA 750 Assay Kit and LabChip XT DNA sizing system. The quality and quantity of all libraries were assessed on 2100 Bioanalyzer using High Sensitivity DNA Kit (Agilent Technologies) and by qPCR with Kapa Library Quantification Kit (KapaBiosystems), respectively.
ChIP- Seq samples were sequenced during single 50 bp read run on Illumina HiSeq 2500 system in High Output mode (using TruSeq SR Cluster Kit v3-cBot-HS and TruSeq SBS Kit v3-HS) or Rapid mode (using TruSeq Rapid Duo Sample Loading Kit, TruSeq Rapid SR Cluster Kit and TruSeq Rapid SBS Kit). Base calling, demultiplexing and generation of fastq files was carried on using Illumina RTA (v1.18.42.0) and bcl2fastq (v1.8.4) software.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina HiSeq 2500

Description

processed data file: macs.Pol2-Egf.vs.Pol2.peaks.bed

Data processing

Basecalls performed using Real-Time Analysis (RTA) software
BCL2FASTQ Conversion Software was used to generate fastq files
ChIP-seq reads were aligned to the hg19 genome assembly using bowtie2 version 2.1.0 with parameter -N 1
sam files were converted to bam and technical replicates were merged with samtools
differential binding locations were called with Macs version 1.4.2, default parameters
Genome_build: hg19
Supplementary_files_format_and_content: bed files were generated with Macs 1.4.2

Submission date

Jan 26, 2015

Last update date

May 15, 2019

Contact name

Krzysztof Goryca

Organization name

Uniwersytet Warszawski

Department

CeNT

Street address

Banacha 2c