Ion Torrent Proton sequencing; Transcriptomic analysis by AmpliSeq Ion Torrent to study the involvement of LINC00707 in the regulation of TGFβ target genes in HaCaT cells
Transcriptomic analysis by AmpliSeq Ion Torrent to study the involvement of LINC00707 in the regulation of TGFβ target genes in HaCaT cells
HaCaT cells that were either transfected with a non-targeting shRNA (shC) and treated with TGFβ1 for 24 h (shC+TGFβ) or stably transfected with two different shRNAs targeting LINC00707 (shLINC00707#2 and shLINC00707#3). Cells were transfected at 80% confluence before treatment with 5ng/ml TGFβ. Human immortalized keratinocytes (HaCaT) cells 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 and 100 mg/ml streptomycin (Sigma-Aldrich Sweden AB, Stockholm, Sweden). Cellular RNA from ~250,000 cells was purified with the Nucleospin RNA kit (Macherey-Nagel, Düren, Germany). RNA concentration was measured with ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE). Total RNA for Ampliseq was extracted from 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). The acquired cDNA was amplified using Ion AmpliSeq Transcriptome Human Gene Expression core panel (Life Technologies) and the primer sequences were then partially digested. Then, DNA adaptors (Ion P1 Adaptor and Ion Xpress Barcode Adaptor, Life Technologies) were ligated to the amplicons. Adaptor ligated amplicons were purified using the Agencourt AMPure XP reagent (Beckman Coulter) and eluted in amplification mix (Platinum PCR SuperMix High Fidelity and Library Amplification Primer Mix, Life Technologies) and amplified. Size-selection and purification was conducted using Agencourt AMPure XP reagent (Beckman Coulter).