Illumina HiSeq 2500 sequencing; CAGE transcriptomes on EBV transformed lymphoblastoid cell lines from central European individuals
CAGE transcriptomes on EBV transformed lymphoblastoid cell lines from central European individuals
CAGE_6B_CTG_UCB1200_s
EBV transformed lymphoblastoide cell lines (LCLs) were cultured in conventional medium for LCLs (RPMI 1640, GlutaMAX; Gibco) with 15% fetal bovine serum (Gibco), 50 U/ml penicillin and 50 µg/ml streptomycin (Gibco). While harvesting cells in growing phase (<10^6 cells/ml), culture media were tested for mycoplasma infection (Venor GeM Mycoplasma detection kit, Sigma-Aldrich) prior to proceed with RNA extraction. For each cell lines, nucleus-enriched RNA fraction was isolated from 20 million cells. Cells were first lysed in chilled lysis buffer (0.8 M sucrose, 150 mM KCl, 5 mM MgCl2, 6 mM 2-mercaptoethanol and 0.5% NP-40) and spin for 5 min at 10,000g (4°C). Nuclei pellets were washed twice with lysis buffer before resuspension in TRIzol Reagent (Life Technologies). The RNeasy kit (Qiagen) was used according to the manufacturer's protocol to extract nucleus-enriched RNA fractions. During the RNA purification process, samples were treated with DNase I (TURBO DNA-free kit, Ambion) following manufacturer recommendations. CAGE libraries were prepared from 3 µg of nuclear enriched RNA, following the protocols published by Takahashi H. et al., Nat. Protoc (2012) 7:542-561