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Sample GSM1462977

Query DataSets for GSM1462977

Status

Public on Aug 01, 2014

Title

RNA-seq_598-SKD_3dDox_rep3

Sample type

SRA

Source name

Human breast adenocarcinoma cell-line MCF7

Organism

Homo sapiens

Characteristics

cell line: MCF7
transgene: ZNF : 598-SKD
dox tx: 3 days

Treatment protocol

ATF plasmids were designed and stably integrated into MCF7 cells as described in (Stolzenburg et al. 2012). Stable lines were grown at 30–80% confluency in Dulbecco’s Modified Eagle’s Medium (Corning, Corning, NY) supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen, Life Technologies, Grand Island, NY) and 1% penicillin/streptomycin; cells were selected using 5 µg/ml puromycin (VWR, Radnor, PA) and 200 µg/ml G418 (VWR, Radnor, PA). ATF expression was induced by treatment with media containing 1µg/ml doxycycline (VWR, Radnor, PA) at 0 h, doxycycline media was refreshed at 48 h, and cells were harvested at 72 h. ATF expression was confirmed by hemagglutinin (HA) tag western blot prior to HA ChIP-seq, histone ChIP-seq and RNA-seq analysis.

Growth protocol

Thawing Cells: 1. Thaw vial immediately in 37º C water bath. Keep O ring above the water surface to prevent contamination. Thaw content with slight shake until only small ice is left in vial. It usually takes 1 min. Spray vial with 70% ethanol all over and wipe its surface with clean tissue in the hood. 2. Open the vial and transfer the content to a 15 ml Falcon tube already containing 5 ml of fresh medium. 3. Spin down at 1000 rpm or 200g for 3 to 5 mins at 4º C. Aspirate supernatant. 4. Resuspend cells in fresh medium and transfer to 150mm x 25mm tissue culture dish. 5. Check the cells under microscope. 6. Cells are cultured in CO2 incubator and medium is changed about every 3 days. 7. It usually takes 3 days or more for cells to recovery from freezing. After cell culture reaches 80-85% confluence, subculture is conducted. Subculture ratio is about 1:3 or 1:4. Passaging Cells: 1. Observe cells to see how confluent they are, whether the cells are alive, whether the cells are contaminated, and whether the cells have the correct morphology. After cell culture reaches 80 to 85% confluence, subculture is conducted 2. Remove media from dish 3. Wash 1x with 10 ml of PBS 4. Add 5 ml of Trypsin and trypsinize for 3 min at 37C. Whack hard – you should see the cells coming down. (Important: never overtrypsinize the cells, so work quickly) 5. Add 5 ml of media and use it to rinse the dish to detach the cells off (4 to 5 times) 6. Spin down at 1000rpm for 3 to 5 min at room temperature. Aspirate supernatant 7. Add 15 ml of media to 15ml tube containing cell pellet, and pipette up and down to mix 8. Add 15 ml of media to each new 150mm x 25mm tissue culture dish (we split 1 dish into 3 to 4 dishes) 9. Add 5mls of cell culturecontaining media to each tissue culture dish. Make sure the media covers the entire area of the dish. Put the cells into 37C with 5% CO2.

Extracted molecule

total RNA

Extraction protocol

For HA-tag ChIP-seq, stable MCF7 cell lines were induced using 100 ng/ml doxycycline (Sigma) at 0 h, doxycycline media was refreshed at 48 h, and cells were harvested at 72 h by crosslinking in a final concentration of 1% formaldehyde. Crosslinking was stopped after 5 min by adding glycine to a final concentration of 125 mM. Crosslinked cells were washed in cold phosphate buffered saline, lysed using 1 ml low-salt IP buffer (150 mM NaCl, 50 mM Tris-HCl (pH7.5), 5 mM EDTA, NP-40 (0.5%), Triton X-100 (1%) containing protease inhibitors) and aliquoted at 1 × 10∧7 cells/ml.
For Bisufite-seq, 2 microgram of genomic DNA was isolated using a QIAeasy DNA mini kit (Qiagen, Venlo, The Netherlands) and sonicated using a Covaris to an average molecular weight of 150 bp. Achievement of the desired size range was verified by Bioanalyzer (Agilent) analysis. Fragmented DNA was repaired to generate blunt ends using the END-It kit (Epicentre Biotechnologies, Madison, WI) according to manufacturer’s instructions. Following incubation, the treated DNA was purified using AmpureX beads from Agencourt. In general, magnetic beads were employed for all nucleic acid purifications in the following protocol. Following end repair, A-tailing was performed using the NEB dA-tailing module according to manufacturer’s instructions (New England Biolabs, Ipswich, MA). Adapters with a 3′ ‘T’ overhang were then ligated to the end-modified DNA. For whole genome bisulfite sequencing, modified Illumina paired-end (PE) adapters were used in which cytosine bases in the adapter are replaced with 5-methylcytosine bases. Depending on the specific application, we utilized either Early Access Methylation Adapter Oligos that do not contain barcodes, or the adapters present in later versions of the Illumina DNA Sample Preparation kits, which contain both indices and methylated cytosines. Ligation was carried out using ultrapure, rapid T4 ligase (Enzymatics, Beverly, MA) according to manufacturer’s instructions. The final product was then purified with magnetic beads to yield an adapter-ligation mix. Prior to bisulfite conversion, bacteriophage lambda DNA that had been through the same library preparation protocol described above to generate adapter-ligation mixes was combined with the genomic sample adapter ligation mix at 0.5% w/w. Adapter-ligation mixes were then bisulfite converted using the Zymo DNA Methylation Gold kit (Zymo Research, Orange, CA) according to the manufacturer’s recommendations. Final modified product was purified by magnetic beads and eluted in a final volume of 20 µl. Amplification of one-half the adapter-ligated library was performed using Kapa HiFi-U Ready Mix for the following protocol: 98º 2′ ; then six cycles of 98º 30′ ′ , 65º 15′′, 72º 60′′; with a final 72º 10′ extension, in a 50 µl total volume reaction. The final library product was examined on the Agilent Bioanalyzer, then quantified using the Kapa Biosystems Library Quantification kit according to manufacturer’s instructions. Optimal concentrations to get the right cluster density were determined empirically but tended to be higher than for non-bisulfite libraries.
RNA was harvested using the Qiagen RNeasy kit and Illumina libraries were made with the Illumina TruSeq Stranded mRNA Sample Prep kit according to the manufacturer’s instructions.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

RNA-seq_598-SKD.txt

Data processing

Illumina Casava1.7.1a5 software used for basecalling.
For ChIP-seq, fastq files were mapped to human genome version GRCh37/hg19 using Bowtie 2.0.1 (34) with default parameters to produce SAM files. SAM files were converted to BAM files using SAMTools (35). BAM files were converted to BED files using BEDTools (36). In order to call ChIP-seq peaks, BED files were submitted individually (each with a similarly mapped MCF7 input BED file as a background reference) to the Sole-Search Tool (37) with the following parameters: permutation number, 5; average chromatin fragment size, 150; alpha value, 0.001; peak merge distance, 0; histone blur, no. Resulting GFF format peak files were sorted by peak height and truncated to the number of peaks in the smallest replicate for each set. The top 40% of peaks for each truncated replicate were tested for statistically significant overlap versus corresponding replicate total peak files using the Sole-Search GFF-overlap Tool. All 40% replicate peak files satisfied the ENCODE standard of 80% overlap with truncated corresponding replicates (http://bit.ly/ 1bAFFGp). Corresponding replicates were then submitted simultaneously as merged sets to the Sole-Search Tool for peak calling, producing sets of merged GFF peak files and SGR visualization files. Again using the Sole-Search GFF-overlap Tool, peaks for each ATF variation that overlapped with pT3-empty-vector GFF files or with regions of aneuploidy in MCF-7 cells were removed to eliminate possible artifacts not resulting from ATF binding. Remaining ATF peak sets were sorted by peak height and plotted against peak rank to select high-confidence (HC) peaks (Supplementary Figures S1A and B), along with visual inspection of SGR format visualization files. The Homer genomic suite of tools (38) was used for tag density plots (annotatePeaks.pl), scatter plots (annotatePeaks.pl), overlap analysis (mergePeaks), genomic locations (annotatePeaks.pl) and de novo motif finding (findMotifsGenome.pl). De novo motif finding for the top 4000 peaks from each HC ATF peak set was performed using all hg19 RefSeq gene promoters as a background for statistical comparison and all nucleotides within the peaks, as called by the Sole-Search tool, were subjected to the analysis. The resulting predominant motif position weight matrix files for each ATF were used to search for the presence of significant motif matches among the top 2500 HC peaks for each ATF. The predominant motif position weight matrix files were restricted to single nucleotide tolerances to represent partial target sequences in order to search the entire genome for instances of partial target sites using annotatePeaks.pl. Histone ChIP-seq enrichment values for all RefSeq promoter regions (TSS ± 1 kb) were calculated from the average tags per bp using annotatePeaks.pl within each promoter region. Tag density plots for histone enrichment at different promoter categories (downregulated, unchanged, upregulated and unbound) were calculated using the -hist 200 option of annotatePeaks.pl. H3K4me3 plots and enrichments before and after induction were compared to ENCODE datasets for H3K4me3 in MCF7 (wgEncodeEH000967) to ensure that uninduced profiles are representative of MCF7 cells.
For Bisulfite-seq, raw reads were mapped with [programs/parameters], and percent methyl-C/C was calculated for every CpG dinucleotide in the human genome. All CpG dinucleotides with a minimum sequencing coverage of 3× were used for downstream analyses. To determine the average DNA methylation surrounding a region of interest, the annotatePeaks tool of the HOMER suite was used with the ‘-ratio’ option and a bin size of 200 bp.
For RNA-seq, prealignment QA/QC and base trimming were performed in Partek Flow v3.0.14.0321 using fastqc and quality trim. Reads were mapped to the Gencode version 19 of the human hg19 reference genome using Tophat (39). Quantification to the transcriptome was performed by Partek expectation maximization (EM). The Partek Flow software suite was made available through the USC Norris Medical Library Bioinformatics Services (http://norris.usc.libguides. com/nml-bioinfo). Genes with a FDR less than 0.05 and a change in expression greater than 2-fold were counted as differentially expressed.
Genome_build: hg19
Supplementary_files_format_and_content: ChIP-seq peak files in gff format, the peaks are called from the merged of all replicated samples
Supplementary_files_format_and_content: Bisulfite-seq Bed files in BED 6+2 format
Supplementary_files_format_and_content: tab-delimited text file with expression values, significance values, FDR values, and statistical model information

Submission date

Jul 31, 2014

Last update date

May 15, 2019

Contact name

Matt Grimmer

E-mail(s)

grimmem1@gene.com

Organization name

Genentech

Department

Oncology Bioinformatics