GSM9290492: NCIH661_siDPPA24_DKD_R1; Homo sapiens; RNA-Seq
RNA extraction was performed on cell pellets collected from each cell line, using the Monarch Total RNA miniprep kit (NEB #T2010S) with DNAse I treatment according to the manufacturers instructions. RNA was quantified using the Qubit™ RNA High Sensitivity (HS) Assay Kit (Invitrogen) on a Qubit 3.0 flourometer. Following RNA quantitation, 1 microgram of RNA was used as input for PolyA+ directional RNA-seq library preparation using the NEBNext Ultra II Directional RNA-seq Kit (#E7765, NEB) with the PolyA mRNA magnetic isolation module (#E7490, NEB) according to the manufacturers instructions. Following adaptor ligation, the final PCR used to incorportate multiplexing oligos for Illumina libraries using the NEBNext Multiplex Oligos for Illumina (Index Primers Set 1-4) was ran for a total of 8 cycles. Individual and pooled libraries were quantified and quality controlled using the dsDNA high sensitivity (HS) Qubit assay (Invitrogen) on the Qubit 3.0 fluorometer (Invitrogen) and D1000 High Sensitivity DNA kit (Agilent) on the TapeStation 2200 (Agilent).The pooled library was sequenced on an Illumina NextSeq 500 or NextSeq 2000 either as single-end 75bp or 100bp configurations respectively.