Computational protocol: Amplification of WHSC1L1 regulates expression and estrogen‐independent activation of ERα in SUM‐44 breast cancer cells and is associated with ERα over‐expression in breast cancer

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Protocol publication

[…] The quality of mRNA was measured using an Agilent Bioanalyzer. All samples had a minimum RNA integrity (RIN) score of 8. Sample labeling was performed using 500 ng of total RNA along with the Target 1‐Round Aminoallyl‐aRNA Amplification Kit 101 (Epicentre, Madison, WI) and Agilent spike‐in controls to produce aminoallyl‐aRNA according to the vendor's protocol. Five μg of each aminoallyl‐aRNA sample and Alexa Fluor 555 or Alexa Fluor 647 (Molecular probes/Life Technologies, Foster City, CA) were used for labeling. Samples were incubated with the dye for 30 min at room temperature. After incubation, samples were run through RNeasy Mini Elute Cleanup columns (Qiagen, Valencia, CA) to remove unincorporated dye. Sample concentration and dye incorporation were then checked on a NanoDrop. Labeled samples were prepared for hybridization following Agilent's “Two‐Color Microarray‐Based GE Analysis” protocol. For microarray hybridization, 825 ng of Alexa Fluor 555 labeled aminoallyl‐aRNA and 825 ng of Alexa Fluor 647 labeled aminoallyl‐aRNA were mixed and allowed to co‐hybridize on the Agilent 60‐mer oligo array (Human Gene Expression V.2, 4X44K) for 17 h at 65°C at 10 rpm in a hybridization oven. Slides were washed with Agilent GE Wash Buffers following Agilent's protocol.Slides were immediately scanned with the Agilent dual laser scanner with SureScan High Resolution Technology. Tiff images were analyzed using Agilent feature extraction software version 10.7.1.1 and protocol GE2_107_Sep09 to obtain fluorescence intensities for each spot on the array. Data were normalized using the linear and lowess method. Normalized data were imported into Genespring for analysis. Statistical significance of differentially expressed genes was determined using four replicate measurements for each probe (gene). A t‐test against zero was performed with normalized log ratios reflecting the change in gene expression (44/NS) The Benjamini and Hochberg multiple test correction was used to determine the false discovery rate. [...] For ChIP‐Seq analysis of ERα binding sites was performed following the procedures originally developed by Carroll, Brown and co‐workers (Carroll et al., , ) and personal communications). Cells were cultured in 150 mm plates to ∼90% confluence (approximately 1 × 108 cells). Cells were treated with 2 mM disuccinyl‐glutamate in PBS for 30 min at room temperature and then treated with 1% fresh formaldehyde for 2.5 min before quenching with 125 mM glycine for 5 min. Cells were washed with cold PBS, and chromatin was prepared using the Covaris High‐Cell SDS chromatin shearing kit with SDS buffer (Cat. #520076) according to the manufacturer's recommended protocol. Chromatin was then sheared with a Covaris S220 sonicator using standard settings for 8 min. Chromatin preparation was optimized for SUM‐44 cells. ChIP was performed using the Magna ChIP A/G ChIP Kit (17‐10085) with an antibody cocktail containing equal concentrations of the ERα antibodies from Thermo Scientific and Santa Cruz. Sequencing libraries from ERα ChIP DNA were prepared with Rubicon ThruPLEX library preparation kits and sequenced as 35 bp single‐end reads on an Illumina Hi‐Seq 2500. Libraries for three biological replicates were prepared and sequenced for each sample type. Read data for each sample was aligned with Bowtie2, and peaks were called with both the MACS version 2 and HOMER FindPeaks peak callers. To generate consensus peak sets for downstream analysis, the called peak sets from each peak caller for each sample were input into DiffBind, which created consensus peak sets consisting of peaks present in at least two replicates for each sample type. These consensus peak sets were used for downstream analysis, including differential occupancy and differential binding. […]

Pipeline specifications

Software tools Bowtie2, HOMER, DiffBind
Application ChIP-seq analysis
Organisms Homo sapiens
Diseases Breast Neoplasms
Chemicals Estradiol, Estrogens