Illumina HiSeq 2000 paired end sequencing; RNA-seq of coding RNAs from protrusions of MDA-MB231 cells treated with control or exosome core complex siRNA
RNA-seq of coding RNAs from protrusions of MDA-MB231 cells treated with control or exosome core complex siRNA
cells were grown in normal DMEM with 10% FBS in standard tissue culture conditions. Cells were transfected with 10nM final concentration of non-targetting vs EXOSC8 siGENOME smartpool siRNAs, using Lipofectamine RNAiMax according to manufacturer's instructions. 72 hr later, cells were fractionated into protrusion and cell-body fractions using 3 micrometer pore polycarbonate transwell filters (75mm membrane Inserts, corning). Filters were coated with collagen and 8 million cells were then seeded in 8ml serum free DMEM on top of the filter, with 12ml of DMEM containing 10% serum in the bottom. Cells were allowed to form protrusions through the pores for 2 hrs in presence of Rho Kinase inhibitor H1152, as it rescues protrusion formation downstream of exosome knockdown. Filters were then washed with PBS. The bottom side (protrusions) of the filter was then lysed in RLT buffer from Qiagen's RNeasy Mini Kit. RNeasy Mini Kit (Qiagen) was used for RNA purification. total RNA quality was assessed using the 2100 Bioanalyzer (Agilent). In case of DNA contamination, DNA levels were first estimated by RT-PCR. If DNA contamination was above 2%, the sample was cleaned up using RNeasy Plus Micro Kit (Qiagen). Poly-A mRNA (1-5% of total RNA) was then isolated from the total RNA sample using the Dynabeads mRNA Purification Kit (Life Technologies). Purified mRNAs were fragmented using the mRNA Fragmentation Reagents (Life Technologies) and ethanol precipitated. The size range of generated fragments was kept at 60 to 1000 nucleotides, with a peak at around 150 nucleotides. To make cDNA from the fragmented RNA samples, NEBNext mRNA Library Prep Reagent Set for Illumina was used (NEB), with the synthesis being performed Strand-wise (first-strand synthesis then second-strand synthesis) followed by purification using QIAquick Columns (Qiagen). End Repair, dA-tailing, and Adapter ligation were all performed based on NEBNext mRNA Library Prep Reagent Set for Illumina instructions. Agarose gel separation was used to select for the appropriate library size. To get a library size of ?400bp (insert size ?330bp) a 350-500bp slice was taken from each of the samples. Gel extraction was then performed using Qiagen MinElute gel extraction kit (Qiagen). To enrich for the correctly adapter-ligated and size-selected cDNA, a PCR was performed using the Herculase II polymerase enzyme (Agilent), also adding the specific indexes for multiplexing. The PCR was then followed by an AMPure bead purification to exclude primer-dimers and other smaller fragments from the final library. The quantity and quality of the prepared library was evaluated on Bioanalyzer and the samples were quantified using qPCR. Subsequently, libraries were combined for multiplexed sequencing runs.