title: GSM2266102: EGF20; Homo sapiens; ChIP-Seq
library_construction_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; 6963), 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.
attributes.tag: GEO Accession
attributes.value: GSM2266102