GSM1556076: DYRK1A Cycling T98G; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
TFs and others
Antigen
DYRK1A
Cell type
Cell type Class
Neural
Cell type
T98G
Primary Tissue
Brain
Tissue Diagnosis
Glioblastoma
Attributes by original data submitter
Sample
source_name
Brain
cell line
T98G
tissue origin
Brain
cell type
Glioblastoma
chip antibody
DYRK1A antibody (ab69811)
chip antibody vendor
Abcam
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Formaldehyde was added to the culture medium to a final concentration of 1% for 10 min at RT and the crosslinking was then quenched with 0.125 M glycine for 5 min. Crosslinked cells were washed twice with TBS, resuspended in 1 ml of Buffer I (5 mM PIPES [pH 8.0], 85 mM KCl, 0.5% NP-40 plus PIC) and incubated on ice for 10 min. Cells were centrifuged at 800 x g for 5 min, and the cell pellet was resuspended in 1.2 ml of Buffer II (1% SDS, 10 mM EDTA, 50 mM Tris-HCl [pH 8.0], 1 mM phenylmethylsulfonyl fluoride and PIC) and incubated for a further 10 min on ice. Samples were centrifuged and the cell pellet was resuspended in 1.2 ml of IP Buffer (100 mM Tris-HCl [pH 8.6], 0.3% SDS, 1.7% Triton X-100 and 5 mM EDTA) prior to sonicating the chromatin to an average size of 200-500 bp (Bioruptor, Diagenode). Extracts (600 μg of total protein) were diluted in 1 ml of IP Buffer and immunoprecipitated overnight at 4°C with the corresponding antibodies (listed in the Supplemental Tables for Extended Experimental Procedures) or control IgG antibodies. Immune complexes were recovered by incubating for 4 h at 4°C with protein G-Sepharose beads (40 ml), and the beads were then washed with three successive 1 ml washes in Low salt Buffer III (50 mM HEPES [pH 7.5], 140 mM NaCl, 1% Triton X-100 plus PIC), and one wash with High salt Buffer IV (50 mM HEPES [pH 7.5], 500 mM NaCl, 1% Triton X-100 plus PIC). Libraries were prepared using the NEBNext® ChIP-Seq Library Prep Reagent Set for Illumina® kit (ref. E6200S) according to the manufacturer's protocol. Briefly, 10 ng of input and ChIP enriched DNA were subjected to end repair, addition of “A” bases to 3′ ends and ligation of SE adapters. All purification steps were performed using Qiagen PCR purification columns (refs. 50928106 and 50928006). Library size selection was done with 2% low-range agarose gels. Fragments with average insert size of 280 bp were cut from the gel, and DNA was extracted using QIAquick Gel extraction kit (ref. 50928706, Qiagen) and eluted in 36 µl EB. Library amplification was performed by PCR on the size selected fragments using the following primers: PCR primer 1.1: 5’-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’ PCR primer 2.1: 5’-CAAGCAGAAGACGGCATACGAGCTCTTCCGATCT-3’ . Final libraries were analyzed using Agilent DNA 1000 chip to estimate the quantity and check size distribution, and were then quantified by qPCR using the KAPA Library Quantification Kit (ref. KK4835, KapaBiosystems) prior to amplification with Illumina’s Cluster Station. Libraries were loaded at a concentration of 7 pM onto the flowcell, and were sequenced 1 x 36 on Illumina’s Genome Analyzer IIx.