GSM5242679: FlpInTREx_control_RNA-seq_rep2; Homo sapiens; RNA-Seq
RNA was isolated from 3 x 10^6 cells with the RNeasy Mini Kit (Qiagen, 74106) following the manufacturer's instructions with the following alterations. Cells were homogenized in 600 μL RLT buffer (with 2-Mercaptoethanol) and passed through a QIAshredder column (Qiagen, 79656) by centrifugation in a table top centrifuge for 2 min at full speed at room temperature. The optional step after the second wash with RPE buffer was applied to dry the membrane. For DNA digestion, RNA was eluted with 85 μL H2O and mixed with 10 μL10 x DNAase buffer and 5 μL DNAse I (NEB, M0303S), then incubated at room temperature for 20 min. After incubation, RNA was purified by following the 'RNA Cleanup' protocol in the RNeasy Mini Handbook. RNA was finally eluted in 50 μL H2O and concentration determined with a NanoDrop 8000 Spectrophotometer. RNA was quantified using the Quant-iT RiboGreen RNA Assay Kit (Thermo Fisher Scientific, R11490) and quality-checked with the RNA 6000 Nano Kit (Agilent, 5067–1511) on a 2100 Bioanalyzer (Agilent, G2939BA) or High Sensitivity RNA ScreenTape Assay (Agilent, 5067–5579, 5067–5580, 5067–5581) on a 4200 TapeStation (Agilent, G2991AA) prior to library generation. Libraries were prepared from total RNA with the TruSeq Stranded Total RNA Library Prep Gold Kit (Illumina, 20020599) according to the manufacturer's instructions. Libraries were analyzed for insert size distribution with the High Sensitivity DNA Kit (Agilent, 5067–4626) on a 2100 Bioanalyzer or the High Sensitivity D1000 ScreenTape Assay (Agilent, 5067–5584, 5067–5585, 5067–5587, 5067–5603) on a 4200 TapeStation. Libraries were quantified using the Quant-iT PicoGreen dsDNA Assay (Thermo Fisher Scientific, P11496).
GEO Accession
GSM5242679