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Sample GSM1287870 Query DataSets for GSM1287870
Status Public on Feb 11, 2014
Title U343 RIPSeq control transfection Rep2
Sample type SRA
Source name U343 cells
Organism Homo sapiens
Characteristics transfection: control
cell line: U343
Treatment protocol MicroRNA mimics were transfected into cells in 10cm dishes using Invitrogen Lipofectamine RNAiMax reagent by reverse transfection according to the manufacturer’s instructions. Transfection complexes were prepared and added directly to the cell at a final concentration of 25nM.
Growth protocol U251 and U343 were grown in DMEM media supplemented with 10% fetal bovine serum in standard cell culture conditions (5% carbon dioxide) and incubator.
Extracted molecule total RNA
Extraction protocol For RNAseq experiments, cells were harvested after 48 hours for total RNA extraction using TRIzol. For RIPSeq, cell extracts were prepared from U251 and U343 transfected cells (miR-137 and control mimics) in polysomal lysis buffer, diluted in NT2 buffer and incubated for 3 hours with protein A-sepharose 4 Fast flow from GE conjugated with a mix of PABP antibodies. Beads were washed in NT2 buffer five times and subsequently digested with Proteinase K. RNA was extracted with phenol-chloroform, precipitated with isopropanol and kept at -80 degrees Celsius until usage.
Total RNA was purified from control and miR-137 transfected cells with Trizol. Total RNA and RIP samples were depleted of 28S, 18S, 5.8S and 5S rRNA using RiboZero rRNA removal kit from Epicentre as per manufacters instructions. The rRNA depleted samples from U251 cells (16μl) were then mixed with 1μL random hexamers (500ng) and 1μL 10X MMLV Buffer, heated to 65oC for 2 minutes and placed on ice. To this mixture was added 2.5mM DTT, 0.5 mM dNTPs, 40 units of Riboguard and 50 units of EpiScript RT. First strand cDNA was then synthesized by incubation for 10 minutes at 25oC, 30 minutes at 42oC and 15 minutes at 70oC, then held at 4oC in a thermocycler with a heated lid. Second strand cDNA was synthesized by addition of 30 mM Tris pH 7.5, 5 mM MgCl2, 5 mM DTT, 0.3 mM each dNTP, 2.5 units of E. coli RNase H and 20 units of E. coli DNA polymerase I. The mixtures were then incubated for one hour at 16oC, followed by 15 minutes at 80oC. The reactions were then purified using Zymo DNA Clean kit and the samples were eluted with 16μL of nuclease-free water. The double-stranded cDNA was then fragmented and tagged using the Nextera DNA library kit from Epicentre as per the protocol supplied by the manufacturer and PCR amplified for 9 cycles. The samples were purified using Zymo DNA Clean kit and quantitated by using a bioanalyzer and Qubit methods, then sequenced on an Illumina GAIIx instrument for 36 cycles. After Ribo-Zero treating the RNA samples from the U343 cells, 50 ng were converted into double-stranded cDNA using the TotalScript cDNA kit as per the supplied protocol and purified using the Zymo DNA Clean and Concentrator kit. The cDNA was then tagmented and amplified using the Nextera DNA sample prep kit and purified using the Zymo DNA clean kit. The samples were quantitated by bioanalyzer and Qubit methods, then sequenced on an Illumina GAIIx instrument for 36 cycles.
Library strategy OTHER
Library source transcriptomic
Library selection other
Instrument model Illumina Genome Analyzer IIx
Description library strategy: RIP-Seq
Data processing Read alignment performed using RMAP
Read counts obtained against a transcriptomic reference created from RefSeq by collapsing isoforms and producing one super-transcript per gene; in-house software used
Genome_build: hg18
Supplementary_files_format_and_content: BED file. Read counts per gene
Submission date Dec 11, 2013
Last update date May 15, 2019
Contact name Philip James Uren
Organization name University of Southern California
Street address 1050 Childs Way, RRI201
City Los Angeles
State/province CA
ZIP/Postal code 90089-2910
Country USA
Platform ID GPL10999
Series (1)
GSE53220 Genomic analyses reveal miR-137 broad impact on genes associated with malignant transformation and neuronal differentiation in glioblastoma cells
BioSample SAMN02440319
SRA SRX390237

Supplementary file Size Download File type/resource
GSM1287870_U343_RIPSeq_control_2_counts.bed.gz 315.0 Kb (ftp)(http) BED
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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