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Sample GSM1552215 Query DataSets for GSM1552215
Status Public on Mar 01, 2015
Title Gene expression si_GFP BioRep1_seq
Sample type SRA
Source name HeLa cell_siGFP_expression
Organism Homo sapiens
Characteristics cell line: HeLa
Treatment protocol mature miR-191 duplex or control siRNA against GFP duplex was transfected with lipofectamine 2000 at a concentration of 100nM in the well
Growth protocol DMEM supplemented with 10% FBS
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.
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 2500
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 miR-191 transfection to control transfection
Submission date Nov 21, 2014
Last update date May 15, 2019
Contact name Vishy Iyer
Phone 5122327833
Organization name University of Texas at Austin
Department Molecular Biosciences
Street address 2500 Speedway Dr. MBB 3.212
City Austin
State/province TX
ZIP/Postal code 78712
Country USA
Platform ID GPL16791
Series (2)
GSE63555 miR-191 regulates human cell proliferation and directly targets multiple oncogenes [seq]
GSE63556 Genome wide miR-191 target profile determined by RIP and gene expression profiling
BioSample SAMN03216704
SRA SRX765671

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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