|
| Status |
Public on Oct 04, 2018 |
| Title |
Input_shNONT (b) |
| Sample type |
SRA |
| |
|
| Source name |
MCF7 cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: MCF7
cell type: Breast cancer cell
passages: Low passages (6-10)
shRNA: Non-targeting
chip antibody: none
|
| Treatment protocol |
Cells were grown in phenored free DMEM and 5% of stripped FBS for 96 hours. Then the cells were treated with E2 for either 45 minutes (ChIP-seq) or 4 hours (RNA-seq).
|
| Growth protocol |
Cell medium was composed as follows: DMEM 1X (Thermo Fisher), 10% serum (Corning), 1X glutamine (Life Technologies), 1X penicillin/streptomycin (Life Technologies). Cells were grown in a CO2 incubator (5% CO2) at 37˚C.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
RNA was extracted using QIAGEN RNeasy kit. DNA after ChIP protocol was isolated using QIAGEN PCR purification kit.
ChIP-sequencing libraries were prepared using the KAPA HTP Library Preparation Kit complemented with NEXTflex DNA Barcodes from Bioo Scientific. 10 ng of DNA was used as starting material for input and ip samples. Libraries were amplified using 13 cycles on the thermocycler. Post amplification libraries were size selected at 250- 450bp in length using Agencourt AMPure XP beads from Beckman Coulter. Libraries were validated using the Agilent High Sensitivity DNA Kit. RNA-sequencing libraries were prepared using the Illumina TruSeq Stranded Total RNA Preparation Kit with Ribo-Depletion. Input RNA quality was validated using the Agilent RNA 6000 Nano Kit. 200ng-1 µg of total RNA was used as starting material. Libraries were validated using the Agilent DNA 1000 Kit. For Bisulfite-seq, genomic DNA was quantified with a Qubit 3.0 instrument. ~250 ng of genomic DNA was then digested with the restriction endonuclease MspI (New England BioLabs). Resulting fragments underwent size selection for fragments ~100-250-bp in length using SPRI beads (MagBio Genomics) and subsequent bisulfite conversion using the EZ DNA Methylation-Lightning Kit (Zymo Research) per the manufacturer’s protocol. Bisulfite conversion efficiency averaged > 99% as estimated by the measured percent of unmethylated CpGs in λ-bacteriophage DNA (New England BioLabs N3013S) added at a 1:200 ratio to each sample. Libraries for Illumina-based sequencing were prepared with the Pico Methyl-Seq Library Prep Kit (Zymo Research) using Illumina TruSeq DNA methylation barcodes. Libraries were run on a High Sensitivity chip using an Agilent TapeStation 4200 to assess size distribution and overall quality of the amplified library. Fluorometric quantitation and TapeStation size distribution estimates permitted equimolar pooling, and 6 pooled libraries per run were sequenced on an Illumina NextSeq 500 instrument using the NextSeq 500/550 V2 High Output reagent kit (1 x 75 cycles).
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
Cells treated with non-targeting shRNA.
Fragmented genomic DNA
|
| Data processing |
Basecalls performed using Illumina RTA2 software.
Indexed samples were demultiplexed to fastq files with bcl2fastq v2.17.1.14.
Both ChIP-seq and RNA-seq reads were trimmed from 3' end until the final base had a quality score > 30, using Trimmomatic v0.33, discarding reads left with < 20 bp.
Bisulfite-seq reads were trimmed of 10 base pairs from the 5’ end with TrimGalore! v0.4.3 to remove primer and adapter sequences.
ChIP-seq reads were aligned to UCSC hg19 using Bowtie version 1.1.2. Only uniquely mapping reads with at most two mismatches were retained.
RNA-seq reads were aligned to UCSC hg19 using Tophat version 2.1.0. Only uniquely mapping reads with at most two mismatches were retained.
Bisulfite-seq reads were aligned to UCSC hg19 and methylation extraction ignoring 1 base at the 3’ end (after reviewing the M-bias plots) were performed with Bismark v0.16.3.
To create ChIP-seq coverage plots, the locations of the mapped ChIP-seq reads were extended to 150 bp to represent sequenced fragments, renormalized (to reads per million, rpm) and reformatted in the bigWig file format.
To create RNA-seq coverage plots, the mapped RNA-seq reads were split according to strand, renormalized (to reads per million, rpm) and reformatted in the bigWig file format.
Genome_build: hg19 (GRCh37)
Supplementary_files_format_and_content: bigWig files were generated using the genomicRanges R package. Score represents the normalized coverage of RNA or ChIP fragments at a given genomic coordinate.
Supplementary_files_format_and_content: Bismark tab-delimited "coverage" files for cytosines in CpG context with the following columns: <chromosome> <start position> <end position> <methylation percentage> <count methylated> <count unmethylated>
|
| |
|
| Submission date |
Oct 02, 2018 |
| Last update date |
Oct 04, 2018 |
| Contact name |
Ali Shilatifard |
| E-mail(s) |
[email protected]
|
| Organization name |
Northwestern University Feinberg School of Medicine
|
| Department |
Department of Biochemistry and Molecular Genetics
|
| Lab |
Shilatifard Lab
|
| Street address |
320 E Superior St
|
| City |
Chicago |
| State/province |
IL |
| ZIP/Postal code |
60611 |
| Country |
USA |
| |
|
| Platform ID |
GPL18573 |
| Series (1) |
| GSE120756 |
TET2 coactivates gene expression through demethylation of enhancers |
|
| Relations |
| BioSample |
SAMN10165340 |
| SRA |
SRX4786569 |
