title: Hs Input plus
organism: Homo sapiens
description: Protocols: Human triple negative breast cancer cells Hs578T were cultured in DMEM supplemented with 10% fetal bovine serum in the presence of 1% penicillin-streptomycin. Cells were starved for 18 h in DMEM with 0% fetal bovine serum and stimulated with TGFβ1 (5 ng/ml final concentration). Cells were cultured using standard conditions and then cells were fixed with 2% formaldehyde for 10 min at 37°C. Immediately, formaldehyde was removed and cells were washed with ice-cold PBS twice. Cells were scraped in PBS and spun down at 4,000 rpm for 5 min Supernatant was removed and cells were lysed with SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8.1) with added protease inhibitor cocktail (Roche diagnostics) for 20 min on ice. Lysed cells were then sonicated for obtaining an average fragment size of 250 bp. Input of about 10 % was aliquoted and frozen at -20°C. The remaining lysate was then diluted 10 times with ChIP dilution buffer (0.01% SDS, 1.1% Triton-X 100, 1.2 mM EDTA, 16.7 mM Tris-HCl pH 8.1, 167 mM NaCl) with protease inhibitors, and proceeded for immnoprecipitation using anti-ZEB1 (Santa Cruz Biotech. SC-25388) antibodies or a rabbit antiserum, overnight at 4°C. Protein A dynabeads (Novex, 10001D) were added to this lysate and incubated for 2 h at 4°C. Beads were then washed once with low salt buffer (0.1% SDS, 1% Triton-X 100, 2 mM EDTA, 20 mM Tris-HCl pH 8.1, 150 mM NaCl), once with high salt buffer (0.1% SDS, 1% Triton-X 100, 2 mM EDTA, 20 mM Tris-HCl pH 8.1, 500 mM NaCl), once with lithium chloride wash buffer (0.25M LiCl, 1% IGEPAL, 1% deoxycholic acid, 1 mM EDTA, 10 mM Tris-HCl pH 8.1), and then twice with TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0). After these series of washing steps, the beads and the Input samples were then re-suspended in elution buffer (1% SDS, 1 mM NaHCO3) and mixed up and down for 30 min and left for de-crosslinking in the presence of 0.2 mM NaCl at 65°C overnight. The eluted chromatin was then subjected to proteinase K digestion followed by phenol-chloroform extraction. The respective input was then used to normalize the DNA in each sample that was subjected to chromatin immunoprecipitation. The extracted DNA was then subjected to either PCR quantification or sequencing experiments. ChIP DNA was obtained with 4 biological and technical replicates and pooled for sequencing. The quality of the ChIP DNA obtained was analyzed with a bioanalyzer. The DNA obtained from Input and ChIP were sheared by sonication using the Covaris S2 instrument (Covaris, Inc.). ChIP-seq libraries were constructed from the sheared samples using AB Library Builder System (Life Technologies), followed by amplification and ''Wildfire” conversion (massive parallel monoclonal template generation, www.thermofisher.com), according to the manufacturer's protocols. Sequencing was performed at 75 bp read length on the SOLiD 5500W system (Life Technologies). Library preparation was performed using library kit (5500 SOLiD Library Builder Fragment Core Kit + 5500W Conversion Primer Kit), after which sequencing was performed using the sequencing machine (SOLiD 5500W) at sequencing unit (SOLiD5500W FlowChip).
attributes.tag: ENA first public
attributes.value: 2018-04-25
attributes.tag: ENA last update
attributes.value: 2016-11-17
attributes.tag: External Id
attributes.value: SAMEA4553346
attributes.tag: INSDC center alias
attributes.value: Ludwig Cancer Research and Institute of medical biochemistry and microbiology, SciLife lab, Uppsala University, Sweden.
attributes.tag: INSDC center name
attributes.value: Ludwig Cancer Research and Institute of medical biochemistry and microbiology, SciLife lab, Uppsala University, Sweden.
attributes.tag: INSDC first public
attributes.value: 2018-04-25T17:02:54Z
attributes.tag: INSDC last update
attributes.value: 2016-11-17T11:59:11Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-5241:Hs Input plus
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell line
attributes.value: HS578T
attributes.tag: cell type
attributes.value: epithelial cell
attributes.tag: common name
attributes.value: human
attributes.tag: disease
attributes.value: breast carcinoma
attributes.tag: sample name
attributes.value: E-MTAB-5241:Hs Input plus