GSM7440255: HeLa cells, mutated gdf6Y (del9), rep5; Homo sapiens; RNA-Seq
To isolate RNA from transfected cells, the QIAGEN RNeasy Mini Kit was used. Cells were lysed directly in the wells using RT buffer containing ß-mercaptoethanol. Samples were further homogenized using QIAshredder (QIAGEN). DNase digestion was performed on-column using the RNase-Free DNase Set (QIAGEN) as suggested in the RNeasy Mini Kit protocol. The RNA was eluted in 50 µl DEPC-treated H2O. Libraries were prepared from 300 ng of input material (total RNA) using NEBNext Ultra II Directional RNA Library Preparation Kit in combination with NEBNext Poly(A) mRNA Magnetic Isolation Module and NEBNext Multiplex Oligos for Illumina (Unique Dual Index UMI Adaptors RNA) following the manufacturer's instructions (New?England Biolabs). Deviating from the instruction, in order to avoid over-amplification of libraries in the final amplification step, qPCR was performed for each library priorily for determine the optimal cycle number (exponential phase) .