|
| Status |
Public on Dec 21, 2015 |
| Title |
tnfpreUV2 |
| Sample type |
SRA |
| |
|
| Source name |
Foreskin fibroblasts (HF1)
|
| Organism |
Homo sapiens |
| Characteristics |
uv exposure: 20 J/m^2
time between uv exposure and bru labeling: 0 hours
bru labeling time: 0.5 hours
time between bru labeling and rna extraction: 0 hours
time between uv exposure and rna extraction: 0.5 hours
labeling agent: Bromouridine (Bru)
|
| Treatment protocol |
Cells were washed in PBS and irradiated in 100 ul PBS on 100 mm plates with a UVC lamp producing 1 J/m2/s. Cells were then incubated in conditioned media containing 2 mM bromouridine (BrU) at 37C for a 30 min.
|
| Growth protocol |
HF1 cells were grown as monolayers in MEM supplied with 10% fetal bovine serum and antibiotics (Invitrogen). K562 cells were grown in suspension in IMDM with 10% FBS. All cells were maintained at 37 C in a humidified 5% CO2 atmosphere.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated by using TRIzol reagent (Invitrogen), and Bru-labeled RNA was isolated from the total RNA by incubation with anti-BrdU antibodies (BD Biosciences) conjugated to magnetic beads (Dynabeads, Goat anti-Mouse IgG; Invitrogen) under gentle agitation at room temperature for 1 h. For more detail, see Paulsen et. al 2013
Bru-labeled RNA was mixed with first strand buffer and random primers and fragmented by heating at 85 ̊C for 10 minutes. The first strand cDNA was then synthesized, in the presence of Actinomycin D to result in strand specific reads (only applicable to certain samples as indicated in sample table above). After purifying the first strand cDNA using AMPure RNAclean beads (Beckman Coulter), the second strand cDNA was synthesized. The resulting cDNA was purified with AMPure XP beads, after which the Illumina TruSeq RNA Sample Prep Kit was used to repair the cDNA ends, adenylate and ligate adaptors to the cDNA. The samples were then run on a 3% agarose gel and size-selected by excising gel slices in the 300bp region. These gel slices were purified using the QIAEX II Gel Extraction Kit (Qiagen) and then the Illumina TruSeq Kit PCR reagents were used to enrich the DNA fragments. After a final purification using AMPure XP beads, the quality and concentration of the DNA libraries were determined using an Agilent Bioanalyzer.
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
molecule: bru-labeled RNA
|
| Data processing |
Library strategy: Bru-Seq
Illumina Casava v1.8.2 software used for basecalling.
Reads were mapped to the Human ribosomal DNA complete repeating unit (U13369.1) using Bowtie v.0.12.8 and parameters -n 3 -k 1 -m 1
Reads that did not map to the ribosomal DNA were mapped to the hg19 genome using TopHat v1.4.1 and parameters –min-isoform-fraction 0, --max-multihits 1, --no-closure-search, --no-coverage-search, --bowtie-n, --initial-read-mismatches 3
A coverage determination was then made for every base in the genome using Bedtools v.2.16.2 such that a base covered by one read was recorded as having a coverage of 1/read_length
Reads Per Kilobase of exon per Megabase of library size (RPKM) were calculated using a protocol from Chepelev et al., Nucleic Acids Research, 2009. In short, exons from all isoforms of a gene were merged to create one meta-transcript. The number of reads falling in the exons of this meta-transcript were counted and normalized by the size of the meta-transcript and by the size of the library.
Genome_build: hg19
Supplementary_files_format_and_content: The columns of the abundance measurement files indicate: (1) chromosome; (2) start; (3) end; (4) name; (5) score; (6) strand; (7) gene length; (8) feature type – exon, intron, etc; (9) number of introns and exons; (10) feautre length; (11) hits count; (12) density – count/length; (13) RPKM - count/(length/1E3)/(mapped read count/1E6)
|
| |
|
| Submission date |
Nov 25, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Mats Ljungman |
| E-mail(s) |
tenbroek@med.umich.edu, bedik@umich.edu, ivenkat@umich.edu
|
| Organization name |
University of Michigan
|
| Street address |
NCRC, B520 Room 1346 2800 Plymouth Rd.
|
| City |
Ann Arbor |
| State/province |
Michigan |
| ZIP/Postal code |
48109-2800 |
| Country |
USA |
| |
|
| Platform ID |
GPL11154 |
| Series (1) |
| GSE75398 |
Identifying transcription start sites and active enhancer elements using BruUV-seq |
|
| Relations |
| BioSample |
SAMN04296162 |
| SRA |
SRX1452346 |
