|
| Status |
Public on Jul 23, 2013 |
| Title |
miCLIP_NSUN2_HEK293_rep6 |
| Sample type |
SRA |
| |
|
| Source name |
miCLIP_NSUN2_HEK293
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HEK293
cell type: Embryonic kidney cells
transfected with: myc-tagged NSUN2 C271A construct
iclip antibody: anti-myc antibody
|
| Treatment protocol |
myc-tagged NSUN2 C271A construct was transfected into HEK293 cells which were harvested 24 hours post-transfection. Cells were then lysed in lysis buffer and treated with high concentration of DNase and low concentration of RNaseI to partially fragment RNAs. Lysates were incubated with proteinG dynabeads in the presence of anti-myc antibody to isolate NSUN2-RNA complexes.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was obtained using the standard Trizol extraction protocol and the mRNA fraction was subsequently purified using oligodT magnetic dynabeads.
Following stringent washing, 3’end dephosphorylation was performed with T4 polynucleotide kinase before addition of a 3’ pre-adenylated linker using RNA ligase. 5’end labelling was then performed using T4 PNK and 32P-ATP before protein-RNA complexes were eluted and run on denaturing gels. Nitrocellulose transfer was then performed and the radioactive signal was then used to dissect nitrocellulose pieces that contained NSUN2-partially digested RNA complexes. RNA was recovered by incubating the nitrocellulose pieces in a buffer containing ProteinaseK and 7M urea. Reverse transcription was performed next, with the oligonucleotides for reverse transcription containing two inversely oriented adaptor regions separated by a BamHI restriction site as well as a barcode region at their 5′ end containing a 5-nt random barcode to enable tracing of individual cDNAs. cDNAs were size-purified on TBE-Urea gels before being circularized by CircLigase II. Circularised cDNAs were then annealed to an oligonucleotide complementary to the BamHI site and then BamHI digested. Linearized cDNAs were then PCR-amplified using primers complementary to the adaptor regions using either 25 or 35 cycles of PCR.
Libraries were prepared using the Illumina TruSeq mRNA library prep kit.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer II |
| |
|
| Description |
Sample 6
|
| Data processing |
Library strategy: iCLIP-seq
Potential PCR duplicates were removed and reads with identical barcodes were counted to determine exact cDNA counts. Adapter and barcodes were removed and reads were aligned to the human reference genome (GRCh37/hg19) using bowtie (0.12.9). Only the reads uniquely aligning to the genome with one mismatch were considered for further analysis.
Genome_build: GRCh37/hg19
Supplementary_files_format_and_content: iCLIP read counts per ncRNAs from ENSEMBL (release 68, October 2012) and tRNA gene loci (GtRNA, October 2012), tab-spaced
|
| |
|
| Submission date |
Feb 19, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Sabine Dietmann |
| Organization name |
Washington University School of Medicine
|
| Street address |
4565 McKinley Avenue
|
| City |
St Louis |
| State/province |
MO |
| ZIP/Postal code |
63110 |
| Country |
USA |
| |
|
| Platform ID |
GPL9115 |
| Series (2) |
| GSE44385 |
NSun2-mediated cytosine-5 methylation in Vault non-coding RNA determines its processing into small RNAs [iCLIP] |
| GSE44386 |
NSun2-mediated cytosine-5 methylation in Vault non-coding RNA determines its processing into small RNAs |
|
| Relations |
| SRA |
SRX242695 |
| BioSample |
SAMN01923027 |
