|
| Status |
Public on Aug 04, 2022 |
| Title |
IgG Cytosol RIP - replicate 2 |
| Sample type |
SRA |
| |
|
| Source name |
MCF cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: MCF-7
antibody: rabbit IgG (Santa Cruz, sc-2027)
genotype: wild type
|
| Treatment protocol |
A total amount of lysate corresponding to 500 ug of protein content from MCF7 and MDA-MB 231 cells were pre-cleaned with protein A/G Plus-Agarose beads (Santa-Cruz, sc-2003) for 1 hour at 4 °C. Five to ten percent of pre-cleaned sample was saved as input for subsequent analysis. The remainder was used in immunoprecipitation reactions with rabbit IgG (Santa Cruz, sc-2027) or rabbit polyclonal antibody against DKC1 (Genetex GTX 109000) see antibody table) with incubation overnight at 4 °C. Afterwards, anti-dyskerin interacting fraction was captured using protein A/G Plus-Agarose beads and washed several times with Wash Buffer (25 mM Tris-HCl pH 7.5, 150 mM KCl, 5 mM MgCl2, 1mM EGTA, 10 % glycerol and protease and RNAse inhibitor cocktail). Two thirds of the immunoprecipitated solution were used for RNA extraction.
|
| Growth protocol |
MCF-7 (female, estrogen-positive invasive breast ductal carcinoma derived) cells were cultured in RPMI 1640 medium supplemented with 10% FBS, 100 U/ml penicillin, 1 mg/ml streptomycin, and 2 mM L-glutamine.
|
| Extracted molecule |
cytoplasmic RNA |
| Extraction protocol |
RNA was purified by extraction with 1 volume of phenol-chloroform and precipitated with isopropanol.
RNA libraries were generated using the SMART-Seq Stranded Kit (Takara), using 10ng of input material for each sample.
|
| |
|
| Library strategy |
RIP-Seq |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
rawcounts_genes.tsv
rawcounts_transcripts.tsv
|
| Data processing |
Illumina Casava 1.8 software was used for basecalling and demultiplexing.
Sequenced reads were trimmed for adaptor sequence and low-quality nucleotides, then quantified against the Gencode v28 transcriptome using STAR (genes) and Salmon(transcripts) with default parameters
Genome_build: GRCh38.p7
Supplementary_files_format_and_content: tab-delimited text files include raw read counts for each Sample
|
| |
|
| Submission date |
Nov 13, 2020 |
| Last update date |
Aug 04, 2022 |
| Contact name |
Erik Dassi |
| E-mail(s) |
erik.dassi@unitn.it
|
| Organization name |
University of Trento
|
| Department |
CIBIO
|
| Street address |
Via Sommarive, 9
|
| City |
Trento |
| State/province |
TN |
| ZIP/Postal code |
38123 |
| Country |
Italy |
| |
|
| Platform ID |
GPL16791 |
| Series (2) |
| GSE161479 |
The pseudouridine synthase DKC1 binds to cytoplasmic transcripts containing H/ACA-box SnoRNA sequences affecting nuclear hormone receptor dependence [RIP-seq] |
| GSE161481 |
The pseudouridine synthase DKC1 binds to cytoplasmic transcripts containing H/ACA-box SnoRNA sequences affecting nuclear hormone receptor dependence |
|
| Relations |
| BioSample |
SAMN16794632 |
| SRA |
SRX9507672 |
