title: GSM1287858: U251 RNASeq control transfection Rep2; Homo sapiens; RNA-Seq
library_construction_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.
attributes.tag: GEO Accession
attributes.value: GSM1287858