|
| Status |
Public on Apr 21, 2013 |
| Title |
DNMT2-Aza-IP |
| Sample type |
SRA |
| |
|
| Source name |
HeLa cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HeLa
transgene (lentiviral infection): V5-tagged hDNMT2
5-aza-c treatment: 3μM at 18 hrs post infection for 12 hrs
cell harvest time: 30 hrs post infection
ip antibody: Anti-V5 Antibody
ip antibody vendor: Invitrogen
ip antibody catalog #: R960-25
molecule subtype: Fragmented IP-RNA
|
| Treatment protocol |
5-Azacytidine treatment at final concentration of 3μM for 12 hrs (at 18 hrs post infection by lentivral vectors)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
After the last washing round the protein-RNA adducts were subjected to RNA fragmentation on the beads, using RNA fragmentation reagent (Ambion) at 94C for exactly 5min in a thermocycler, followed by 1min incubation on ice and then quenched by fragmentation stop solution (Ambion). The fragmented RNA was then purified by ethanol precipitation followed by Trizol (Invitrogen) RNA extraction and used for library preparation. Illumina’s directional mRNA-Seq sample preparation protocol
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
Sample 1
|
| Data processing |
The “Make Transcriptome” application from open source USeq package (Utah Bioinformatics Shared Resource Center) was used to build the “transcriptome index file” to include the different combinations of the splicing events for genes with multiple exons.
The commercial "Novoalign" package (Novocraft) was used for sequence alignments with options to allow mismatches, reporting 18bp or larger inserts and reporting all the reads mapped to the repeats and generating SAM formatted alignment files.
The “RNASeq” application of the USeq package was used for assessment of the enriched/over-expressed transcripts using SAM files as input.
The “RNASeq” application of the USeq package was used to generate the BAM/BAI files, from SAM alignments input files, for visualization of the mapped reads.
The Integrative genomics viewer (IGV) was used to visualize the alignment files, inspect the mapped reads at base pair resolution and get the base distribution reports at desired locations (e.g C38 position of DNMT2 target tRNAs).
The “get_datasets.pl” script of the Biotoolbox package were used to calculate and compare the total number of mapped reads to each individual tRNA type in DNMT2- and DsRed-Aza-IP datasets.
Genome_build: hg19
Supplementary_files_format_and_content: Each processed data xls file contains detailed description in the first 'Table Legends & Header Info' spreadsheet.
|
| |
|
| Submission date |
Jun 27, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Vahid Khoddami |
| Organization name |
Huntsman Cancer Institute-University of Utah
|
| Department |
Oncological Sciences
|
| Lab |
Bradley R Cairns
|
| Street address |
4350, 2000 Circle of Hope, HCI
|
| City |
SLC |
| State/province |
UT |
| ZIP/Postal code |
84112 |
| Country |
USA |
| |
|
| Platform ID |
GPL11154 |
| Series (1) |
| GSE38957 |
Identification of direct targets and modified bases of RNA cytosine methyltransferases |
|
| Relations |
| SRA |
SRX156242 |
| BioSample |
SAMN01081613 |
