Illumina HiSeq 2500 paired end sequencing; RNA-seq of MCF-7 (breast adenocarcinoma) and 2102Ep (embryonic carcinoma) cells upon LINE-1 knockdown.
RNA-seq of MCF-7 (breast adenocarcinoma) and 2102Ep (embryonic carcinoma) cells upon LINE-1 knockdown.
untreated Whole cell total RNA was purified by a double TRI Reagent extraction (Molecular Research Center), DNase-treatment (TURBO DNase, Life technologies) and a final clean up using the RNeasy mini extraction kit (Qiagen). Poly(A)+ RNA was purified from total RNA using oligo(dT) magnetic beads. Poly(A)+ RNA was isolated from the total RNA samples and fragmented with ultrasound (1 pulse of 15 sec at 4Â°C). First-strand cDNA synthesis was primed with random hexamers. Then, the Illumina TruSeq sequencing adapters were ligated to the 5' and 3' ends of the cDNA. The cDNA was finally amplified with PCR using a proof reading enzyme and between 12 and 14 cycles. The cDNA was purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics). For Illumina sequencing, the cDNA samples were pooled in approximately equimolar amounts. The cDNA pool was size fractionated in the size range of 350-550 bp on a preparative agarose gel.
Experimental Factor: 2102Ep
cell line
Experimental Factor: untreated
RNA interference
Experimental Factor: embryonal carcinoma
disease