|
| Status |
Public on May 31, 2018 |
| Title |
CTCF_siRNA_MCF10A_RNA-seq_2 |
| Sample type |
SRA |
| |
|
| Source name |
Human Mammary Epithelial Cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Transformed nontumorigenic
experiment: RNA-SEQ
treatment: Cells were transfected with 50 nM onTARGETplus SMARTpool CTCF siRNAs (Dharmacon L-020165) at seeding in high confluency using DharmaFECT reagent 1.
|
| Growth protocol |
MCF10A cells were grown in DMEM/F12 supplemented with 20ng/ml of EGF, 0.5mg/ml hydrocortisone, 100ng/ml of cholera toxin, 10mg/ml of insulin and 5% of horse serum.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
1 µg of total RNA sample was used as provided by the user. Sample RNA Integrity Numbers were in the range 9.6-10 when assayed on an Agilent 2100 Bioanalyzer. PolyA+ fraction was purified and randomly fragmented, converted to double stranded cDNA and processed through subsequent enzymatic treatments of end-repair, dA-tailing, and ligation to adapters as in Illumina's "TruSeq Stranded mRNA Sample Preparation Part # 15031047 Rev. D" kit (this kit incorporates dUTP during 2nd strand cDNA synthesis, which implies that only the cDNA strand generated during 1st strand synthesis is eventually sequenced). Adapter-ligated library was completed by PCR with Illumina PE primers (8 cycles). The resulting purified cDNA library was applied to an Illumina flow cell for cluster generation and sequenced on an Illumina instrument (see below) by following manufacturer's protocols.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
ChIP-SEQ processing. Image analysis, per-cycle basecalling and quality score assignment was performed with Illumina Real Time Analysis software. Conversion of Illumina BCL files to bam format was performed with the Illumina2bam tool (Wellcome Trust Sanger Institute - NPG).
ChIP-seq reads were aligned to the hg19 genome assembly using bwa (version 0.6.1-r104) under default parameters. Unmapped reads were removed using samtools (version 1.3.1) running 'samtools view -F 4'. Reads were sorted and replicates removed using picardtools (version 1.60). Peak calling was performed using macs2 (version 2.1.1.20160309) setting following parameters: '-q 0.05' and '-extsize (value obtained from macs2 predictd step)' and using input as the control.
RNA-SEQ processing. Read files were quality-checked with FastQC. Reads were aligned to the human genome (GRCh37/hg19) with TopHat-2.0.10 (using Bowtie 1.0.0 and Samtools 0.1.19), allowing 2 mismatches and 5 multihits. Transcripts quantification and differential expression calculated with Cufflinks 2.2.1. Homo sapiens GRCh37/hg19 transcript annotations from the UCSC Genome Browser were used.
4C-SEQ processing. Read files were feeded to HTSstation following indicated steps: demultiplexing, mapping, 4c. David, FPA et al. HTSstation: A Web Application and Open-Access Libraries for High-Throughput Sequencing Data Analysis. PLoS ONE 9, e85879 (2014). Noordermeer et al., The dynamic architecture of Hox gene clusters, Science, 334:222-225, 2011
Genome_build: GRCh37/hg19
Supplementary_files_format_and_content: bigwig files were generated using ucsc's bedGraphToBigWig tool converting begraph (output of peakcalling step) to bigwig
|
| |
|
| Submission date |
Jul 26, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Ana Losada |
| E-mail(s) |
[email protected]
|
| Phone |
+34 - 917 328 000
|
| Organization name |
Centro Nacional de Investigaciones Oncológicas (CNIO)
|
| Department |
Molecular Oncology Programme
|
| Lab |
Chromosome Dynamics Group
|
| Street address |
C/ Melchor Fernández Almagro, 3.
|
| City |
Madrid |
| State/province |
Madrid |
| ZIP/Postal code |
28029 |
| Country |
Spain |
| |
|
| Platform ID |
GPL11154 |
| Series (1) |
| GSE101921 |
Distinct roles of cohesin-SA1 and cohesin-SA2 in 3D chromosome organization |
|
| Relations |
| BioSample |
SAMN07417368 |
| SRA |
SRX3040170 |
