Charting the functional landscape of phosphorylation through biophysical and multi-omics integration
Charting the functional landscape of phosphorylation through biophysical and multi-omics integration
Approximately 8*10^6 cells were lysed in 600 µl RLT buffer (Qiagen, #74181) and RNA was extracted using the RNeasy 96-well plate kit (Qiagen, #74181) according to manufacturer's instructions including the DNase step.  Approximately 8*10^6 cells were lysed in 600 µl RLT buffer (Qiagen, #74181) and RNA was extracted using the RNeasy 96-well plate kit (Qiagen, #74181) according to manufacturer's instructions including the DNase step.  The initial RNA was QCed using Agilent Bioanalyzer with the RNA Nano Assay kit as per the manufacturer's protocol. The RNA sample set was then standardized to 200 ng total RNA in 50 ul using the concentration values given by the Bioanalyzer. The libraries were prepared on a Beckman Coulter Automated Workstation Biomek i7 Hybrid (MC + Span-8). For library preparation an automated version of the NEBNext® Ultra™ II Directional RNA Library Prep Kit was used, following section 1 - Protocol for use with NEBNext Poly(A) mRNA Magnetic Isolation Module 60. An adaptor dilution of 1 to 30 was used, the samples were individually barcoded using unique dual indices during the PCR using 12 PCR cycles as per the manufacturer's protocol. The individual libraries were quantified using the Qubit HS DNA assay as per the manufacturer's protocol. For the measurement 1ul of sample in 199ul of Qubit working solution was used. The quality and molarity of the libraries was assessed using Agilent Bioanalyzer with the DNA HS Assay kit as per the manufacturer's protocol[v]. The assessed molarity was used to equimolarly combine the individual libraries into one pool for sequencing. 