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| Status |
Public on Jun 15, 2015 |
| Title |
HEK293T_Input |
| Sample type |
SRA |
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| Source name |
HEK293T cells
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| Organism |
Homo sapiens |
| Characteristics |
cell type: Epithelial fetal human kidney cells
cell modifications: Contains SV40 T-antigen
ip antibody: none
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| Treatment protocol |
Three micrograms of nucleic acid sample was then incubated overnight with the S9.6 antibody, following which the RNA:DNA hybrids were enriched by immunomagnetic precipitation using Dynabeads (M-280 Sheep anti-mouse IgG). The sample was then extracted through phenol/chloroform purification, precipitated in the presence of glycogen and re-suspended in EB buffer.
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| Growth protocol |
Both cell lines were grown in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum and 2mM Penicillin-Streptomycin (Invitrogen), at 37°C in 5% CO2. The cells were allowed to reach 80-90% confluence before harvesting with trypsin-EDTA.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Whole cell nucleic acid was isolated through a modified salting out extraction protocol. Nucleic acid was sonicated to an average size of 400-600 bp using the Covaris sonicator. The fragmented nucleic acid was then treated with RNase I (Ambion AM2294) to remove any ssRNA from the sample, phenol/chloroform purified and re-suspended in EB buffer.
Libraries were prepared using elements of a directional RNA-seq protocol. Starting the library preparation at the second strand synthesis step, the RNA of the RNA:DNA hybrid was nicked using RNase H treatment to serve as a primer for the DNA polymerase. The second strand was formed while incorporating dUTP to allow for directional sequencing and the identification of the RNA strand of the RNA:DNA hybrid. Next, the ends of fragments were repaired, adenosine tails added, and Illumina Tru-Seq strand-specific adaptors ligated. UNG treatment was utilized to degrade the dUTP-containing RNA strand of the RNA:DNA hybrid, and barcoded PCR primers were used to amplify the library while maintaining directionality. Libraries were multiplexed and combined for sequencing using Illumina HiSeq 2500 150 bp paired-end sequencing in our institutional Epigenomics Shared Facility.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Fastq files were generated through the Illumina CASAVA pipeline (v1.8). Sequencing reads were then run through the Wasp System (WASP v3.1.5 rev. 6632) hosted pipeline for primary data processing.
The reads were aligned to the hg19 reference genome using Bowtie (v0.12.7), using non-default parameters of --tryhard, -I 50 and -X 650.
Alignments were generated in SAM format, which were then transformed into BAM files using Samtools (version 0.1.8). The aligned sequences in BAM format had PCR duplicates removed, and peaks were called based on input and IP files using MACS v1.4.2.
All peaks containing “N” nucleotides were discarded.
Due to using directional sequencing through the incorporation of dUTP, we were able to determine the RNA-derived sequence of the RNA:DNA hybrids. To do this, we sorted sequencing reads by their bit flag and intersected the reads representing the RNA-derived sequences (which had bit flag 147 or 163) with our RDIP-seq peaks. By assessing the number of RNA reads aligned to the positive or negative reference strand for each peak, we assigned each RDIP-seq peak a “strandedness” value, with +1 being all RNA-derived reads aligned to the positive strand and -1 to the negative strand. Because there were some peaks that had intermediate values of strandedness, we wanted to be confident in assigning the strand to each peak, so we eliminated the middle 10% of strandedness values for peaks (and corresponding peaks) from our dataset.
Genome_build: hg19
Supplementary_files_format_and_content: bed - contains peak chr, start, stop, peak id, reads aligned to pos strand, reads aligned to minus strand, strandedness value, skewing value
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| Submission date |
May 15, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Julie Nadel |
| Organization name |
Albert Einstein College of Medicine
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| Department |
Genetics
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| Lab |
Price 314
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| Street address |
1301 Morris Park Avenue
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| City |
Bronx |
| State/province |
NY |
| ZIP/Postal code |
10461 |
| Country |
USA |
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| Platform ID |
GPL16791 |
| Series (2) |
| GSE68948 |
RNA:DNA hybrids in the human genome have distinctive nucleotide characteristics, chromatin composition, and transcriptional relationships RDIP-seq) |
| GSE68953 |
RNA:DNA hybrids in the human genome have distinctive nucleotide characteristics, chromatin composition, and transcriptional relationships |
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| Relations |
| BioSample |
SAMN03659003 |
| SRA |
SRX1029478 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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