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Sample GSM2856825 Query DataSets for GSM2856825
Status Public on Nov 15, 2017
Title Rao-2017-CMV-OsTIR1-1-T-NS
Sample type SRA
 
Source name Colorectal carcinoma
Organism Homo sapiens
Characteristics cell line: HCT-116-CMV-OsTIR1
treatment: 500uM auxin (360 min)
Treatment protocol Cells were treated with 500uM auxin as described in the characteristics above
Growth protocol Cell lines were cultured according to manufacturer's instructions
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
 
Library strategy OTHER
Library source transcriptomic
Library selection other
Instrument model Illumina HiSeq 2500
 
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
 
Submission date Nov 14, 2017
Last update date May 15, 2019
Contact name Suhas Rao
E-mail(s) suhasrao@post.harvard.edu
Organization name Baylor College of Medicine
Department Molecular and Human Genetics
Lab The Center for Genome Architecture
Street address 1 Baylor Plaza
City Houston
State/province TX
ZIP/Postal code 77030
Country USA
 
Platform ID GPL16791
Series (2)
GSE104334 Cohesin loss eliminates all loop domains
GSE106886 Cohesin loss eliminates all loop domains [PRO-Seq]
Relations
BioSample SAMN08026394
SRA SRX3391668

Supplementary file Size Download File type/resource
GSM2856825_Rao-2017-CMV-OsTIR1-1-T-NS.bam.tdf 348.8 Mb (ftp)(http) TDF
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file
Processed data are available on Series record

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