description: Protocols: HEK293T cells were transfected in T75 flasks with plasmid DNA encoding Vector, 9J10WT, 9J10AMut, GFP-FOXO3a-AAA or GFP, using JetRPIME transfection reagent. Cells were harvested 24 hours post-transfection. Five independent transfections on different days and using cells of different passage number were carried out for each condition. Total RNA was isolated using the Qiagen RNeasy Mini Kit (#74104) complete with DNase digestion (Qiagen #79254), according to manufacturers instructions. RNA was submitted to Novogene for transcriptome sequencing. RNA degradation and contamination was monitored on 1% agarose gels. RNA purity was checked using the NanoPhotometer spectrophotometer (IMPLEN, CA, USA), and RNA integrity and quantitation were assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA). A total amount of 1 μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext UltraTM RNA Library Prep Kit for Illumina (NEB, USA) following manufacturer's recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 150~200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system.
attributes.tag: ENA first public
attributes.value: 2021-05-28
attributes.tag: ENA last update
attributes.value: 2021-05-28
attributes.tag: External Id
attributes.value: SAMEA8142082
attributes.tag: INSDC center alias
attributes.value: MRC Cancer Unit at the University of Cambridge
attributes.tag: INSDC center name
attributes.value: MRC Cancer Unit at the University of Cambridge
attributes.tag: INSDC first public
attributes.value: 2021-05-28T12:15:09Z
attributes.tag: INSDC last update
attributes.value: 2021-05-28T12:15:09Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-10161:GFP-2
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell line
attributes.value: HEK293T
attributes.tag: common name
attributes.value: human
attributes.tag: description of protein/peptide expressed
attributes.value: Green fluorescent protein
attributes.tag: protein/peptide expressed
attributes.value: GFP
attributes.tag: sample name
attributes.value: E-MTAB-10161:GFP-2
attributes.tag: stimulus
attributes.value: Negative control for FOXO3a