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Status |
Public on Feb 04, 2015 |
Title |
Ago2 IP miR-503 BioRep2 |
Sample type |
SRA |
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Source name |
HeLa cell
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Organism |
Homo sapiens |
Characteristics |
cell line: HeLa antibody: Argonaute-2 (AGO2) (ab57113, Abcam) transfected construct: miR-503
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Treatment protocol |
mature miR-503, miR-103, or miR-494 duplex or control siRNA against GFP duplex, or mock transfection (lipofectamine 2000 alone) was transfected with lipofectamine 2000 at a concentration of 100nM in the well
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Growth protocol |
DMEM supplemented with 10% FBS
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Extracted molecule |
total RNA |
Extraction protocol |
Lysates from each sample were split into 2 groups. Group 1: Gene expression samples, total RNA was isolated with TRIzol. Gene 2: Ago2 immunoprecipitation samples, Ago2 was immunoprecipated with an antibody against Ago2 and the associated RNA was isolated with TRIzol. In more detail, 24 hours post transfection, cells were washed 2X with PBS, 0.5 mL of lysis buffer was added to the plate, followed by incubation at 4°C for 30 minutes. Cell lysates were collected by scraping and debris cleared by centrifugation at 14,000 rpm at 4°C. 50 μl of the lysate was collected as input for total RNA profiling. Lysates were pre-cleared by incubating with 50 μl of protein-G beads (Roche) for 1 hour at 4°C and then collecting supernatants (pre-clearing). Pre-cleared lysates were then incubated with 15 μg of antibody against Argonaute-2 (AGO2)(ab57113, Abcam) at 4°C for 3 hours. AGO2 is a protein component of RISC. Following AGO2 antibody incubation, 50 μl of protein-G beads were added to the lysate and incubated for 1 hour at 4°C. Beads were washed 8 times with lysis buffer and RISC-RNA complexes were extracted by adding 1 mL TRIzol reagent (Invitrogen) directly to the beads. RNA extraction with TRIzol was carried out as per the manufacturers instructions. rRNAs were removed using the Ribo-Zero rRNA Removal Kit (Epicentre). The remaining RNA was then fragmented with the NEBNext Magnesium RNA Fragmentation Module (New England BioLabs), and size selected using AMPure XP beads (Agencourt) at 1.8X volume. Ends of the fragmented RNAs were then prepared for adaptor ligation using T4 Polynucleotide Kinase (New England BioLabs). Libraries were prepared from the RNA using the NEBNext Small RNA Library Prep Set according to the manufacturers protocol (New England BioLabs). Libraries were cleaned and further size selected using 0.8X volume AMPure XP beads, and additional size selections with 1.0X beads were performed as necessary.
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Library strategy |
RIP-Seq |
Library source |
transcriptomic |
Library selection |
other |
Instrument model |
Illumina HiSeq 2500 |
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Description |
RNA isolated from Ago2 immunoprecipitation processed data file: Ago2_immunoprecipitation_miR-503_cuffdiff_fpkm.txt
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Data processing |
Illumina CASAVA version 1.8 used for basecalling. Cutadapt was used to trim adaptors, reads mapping to rRNAs and tRNAs were removed, and the remaining reads were mapped to human genome (hg19) by TopHat 2 software version 2.0.9 with parameters -p 6 --b2-fast --library-type=fr-unstranded --no-novel-juncs --no-novel-indels --transcriptome-index Cuffdiff was used to combine biological replicates and assign expression values (FPKM) for each RefSeq gene, run with parameters -p 1 -b -u Genome_build: Hg19 Supplementary_files_format_and_content: tab-delimited text files include FPKM values (value 1 and value 2) from cuffdiff combined biological replicates 1-3 for each treatment group and significance estimates comparing miRNA transfection to control siRNA transfection
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Submission date |
Dec 31, 2014 |
Last update date |
May 15, 2019 |
Contact name |
Vishy Iyer |
E-mail(s) |
iyerlab@gmail.com
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Phone |
5122327833
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Organization name |
University of Texas at Austin
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Department |
Molecular Biosciences
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Street address |
2500 Speedway Dr. MBB 3.212
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City |
Austin |
State/province |
TX |
ZIP/Postal code |
78712 |
Country |
USA |
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Platform ID |
GPL16791 |
Series (2) |
GSE64615 |
hsa-miR-503, hsa-miR-103, and hsa-miR-494 genome wide target profiles [RNA-Seq and RIP-Seq] |
GSE64616 |
Genome-wide hsa-miR-503, hsa-miR-103, and hsa-miR-494 target profiles |
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Relations |
BioSample |
SAMN03274291 |
SRA |
SRX825994 |
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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