title: Sample 11
organism: Homo sapiens
description: Protocols: Transfections of HaCaT cells with plasmid DNA were performed using Lipofectamine 3000 (Invitrogen, Thermofisher Scientific, Stockholm, Sweden). For the generation of stable TGFB2-AS1 over-expressing clones, transfected HaCaT cells were selected in 1.2 mg/ml geneticin (Thermofisher Scientific, Stockholm, Sweden), which was added to the cells 48 h after transfection. The transfected cells were then cultured in the presence of geneticin for two weeks. Then, individual over-expressing clones were selected from the pool of transfected cells, with limiting dilution assay and clones were grown in culture medium, containing geneticin. HaCaT cells expressing pcDNA3 or pcDNA3-TGFB2-AS1 expression vectors were cultured in Dulbecco's modified Eagle's medium (DMEM, Sigma-Aldrich AB, Stockholm, Sweden) supplemented with 10% fetal bovine serum (FBS, Biowest, Almeco A/S, Esbjerg, Denmark) and 100 U/ml penicillin, 100 μg/ml streptomycin (Sigma-Aldrich AB, Stockholm, Sweden). Total RNA from ~250,000 cells was isolated with the Nucleospin RNA kit (Macherey-Nagel, Düren, Germany). RNA concentration was measured with an ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE). Total RNA for Ampliseq was extracted with 3 biological replicates as described above. Fifty ng of RNA was reverse‐transcribed to cDNA using the Ion AmpliSeq™ Transcriptome Human Gene Expression Kit Preparation protocol (Revision A.0, Life Technologies, Stockholm, Sweden). The acquired cDNA was amplified using the Ion AmpliSeq™ Transcriptome Human Gene Expression core panel (Life Technologies, Stockholm, Sweden) and the primer sequences were partially digested. Adaptors (Ion P1 Adapter and Ion Xpress™ Barcode Adapter, Life Technologies, Stockholm, Sweden) were ligated to the amplicons. Adaptor‐ligated amplicons were purified using Agencourt® AMPure® XP reagent (Beckman, Coulter Inc., Brea, CA, USA) and eluted in amplification mix (Platinum® PCR SuperMix High Fidelity and Library Amplification Primer Mix, Life Technologies, Stockholm, Sweden) and amplified. Size‐selection and purification was conducted using Agencourt® AMPure® XP reagent (Beckman, Coulter Inc., Brea, CA, USA).