GSM4971729: Profiling of miRNA from total RNA library from HT29 cells [HT29_1]; Homo sapiens; miRNA-Seq
Tumor samples (PT and LM) were cut into smaller pieces before putting them into the lysing matrix tubes, type "D" (MP Biomedicals). Tubes with 1 ml TRIzol (Invitrogen) were cooled on ice before samples were added. Tumors were homogenized using Fastprep-24-System with 1x 45 sec. @ full speed in TRIzol. After homogenisation, total RNA from tumor samples was isolated according to TRIzol's protocol. Total RNA from HT29 cells was isolated using TRIzol according to TRIzol's protocol without homogenisation steps. In short, an adenylated adapter was ligated to the 3' end of the total RNA by a truncated T4 RNA Ligase 2, followed by the ligation of a 5' RNA-adapter by T4 RNA Ligase 1 (NEB). After reverse-transcription using a specific primer, the cDNA was amplified by PCR, gel-fractionated and bands or regions corresponding to miRNA inserts were cut out. PCR products from these gel slices were eluted, precipitated and dissolved in water. The quality of the libraries was assessed by qPCR and Bioanalyzer measurements.