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Status |
Public on Nov 15, 2017 |
Title |
Rao-2017-CMV-OsTIR1-3-T-S |
Sample type |
SRA |
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Source name |
Colorectal carcinoma
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Organism |
Homo sapiens |
Characteristics |
cell line: HCT-116-CMV-OsTIR1 treatment: 500uM auxin (360 min)
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Treatment protocol |
Cells were treated with 500uM auxin as described in the characteristics above
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Growth protocol |
Cell lines were cultured according to manufacturer's instructions
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Extracted molecule |
total RNA |
Extraction protocol |
To measure changes in transcription resulting from cohesin loss, we performed precision run-on sequencing (PRO-Seq) (Jonkers and Lis, 2015), a variant of global run-on sequencing (GRO-Seq), using a single biotinylated nucleotide (biotin-11-CTP) as previously described (Engreitz et al., 2016). We made one modification to the protocol: at the end of each biotin enrichment, we eluted biotinylated RNAs from the streptavidin-coated magnetic beads by heating beads in 25 μl of 20 mM Tris-HCl pH 7.5, 10 mM EDTA, 2% N-lauroylsarcosine at 95°C for 5 minutes, followed by a magnetic-bead nucleic acid purification with 20 μl of MyONE SILANE beads. During the nuclei preparation step, we processed pairs of RAD21-mAC cells with and without auxin treatment in parallel. In addition, we performed PRO-Seq on HCT-116 CMV-OsTIR1 cells, the parental cell line of RAD21-mAC containing the OsTIR1 gene integrated at the AAVS1 locus and no mAID tags integrated on any protein. standard Illumina library construction protocol, The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the HiSeq 2500 following the manufacturer's protocols
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Library strategy |
OTHER |
Library source |
transcriptomic |
Library selection |
other |
Instrument model |
Illumina HiSeq 2500 |
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Data processing |
Library strategy: PRO-Seq For analysis of PRO-Seq data, we aligned 30-bp paired-end reads to the hg19 reference (bowtie2 v2.1.0, (Langmead and Salzberg, 2012)), removed duplicate reads (Picard http://picard.sourceforge.net), and discarded reads with MAPQ < 30. We counted reads overlapping RefSeq genes (collapsed by gene symbol to the longest isoform) — this quantification procedure includes signal both at the paused position (near the TSS) as well as in the gene body. We identified genes showing significant differences in transcription with DESeq2 (Love et al., 2014), excluding genes with zero coverage in all samples and calling significance at Benjamini-Hochberg corrected p-value < 0.05. Genome_build: hg19 Supplementary_files_format_and_content: counts files, DESeq2 results, tdf files
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Submission date |
Nov 14, 2017 |
Last update date |
May 15, 2019 |
Contact name |
Suhas Rao |
E-mail(s) |
suhasrao@post.harvard.edu
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Organization name |
Baylor College of Medicine
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Department |
Molecular and Human Genetics
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Lab |
The Center for Genome Architecture
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Street address |
1 Baylor Plaza
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City |
Houston |
State/province |
TX |
ZIP/Postal code |
77030 |
Country |
USA |
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Platform ID |
GPL16791 |
Series (2) |
GSE104334 |
Cohesin loss eliminates all loop domains |
GSE106886 |
Cohesin loss eliminates all loop domains [PRO-Seq] |
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Relations |
BioSample |
SAMN08026375 |
SRA |
SRX3391672 |
Supplementary file |
Size |
Download |
File type/resource |
GSM2856829_Rao-2017-CMV-OsTIR1-3-T-S.bam.tdf |
382.3 Mb |
(ftp)(http) |
TDF |
SRA Run Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
Processed data are available on Series record |
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