GSM2561949: GRO Br Set8KO; Mus musculus; OTHER
For library preparation of NRO samples, the Ion Total RNA-Seq Kit v2 for Whole Transcriptome Libraries protocol (ThermoFisher Scientific) was used. Two different sets of NRO samples were prepared:For GRO-EU-seq, liver nuclear RNA from EU-treated mice were prepared by Trizol extraction followed by biotinylation reaction and purification using Click-iT Nascent RNA Capture Kit (Thermo Fisher C10365). 1μl of RNaseIII enzyme and buffer was used to fragment the EU samples, prior to hybridization and ligation, for 2min at 37°C. The samples were further hybridized, ligated and reverse transcribed, according to protocol. GRO-BrU-seq, reactions in isolated liver nuclei were performed as described in (Fang et al., 2014; Cell, Vol 159: pp 1140-1152). After treatment with RppH and polynucleotide kinase, 10μl were directly hybridized with 4μl of Ion adaptor Mix V2 and 6μl hybridization solution, for 10’ at 65°C and 5’ at 30°C followed by ligation with 20μl ligation buffer and 4μl enzyme, for 70’ at 30°C. The samples were then reverse transcribed in a mixture containing 14μl of Ion RT Primer V2 and 8μl 10x Superscript III enzyme. The resulting cDNA from both BR and EU samples was purified with nucleic acid binding beads, eluted in 6μl  ddH2O and amplified according to protocol, in the presence of 45μl Platinum PCR Supermix High Fidelity, 1μl Ion Xpress RNA 3’ Barcode primer and 1μl Ion Xpress RNAseq BC Barcode primer 1-24. [94°C-2’/(97°C-20’’,50°C-30’’,68°C-30’’)*2cycles/(94°C-30’’,62°C-30’’,68°C-30’’)*16cycles/68°C-5’]. The amplified cDNAs were further purified with nucleic acid binding beads, eluted in 15μl ddH2O and their concentration measured in Agilent bioanalyzer. 40 pM of each library was templated using the Ion Chef instrument, loaded on a Ion P1 V3 chip and sequenced on Ion Proton sequencer.
GEO Accession
GSM2561949