GSM3762874: Dnttip1KO1_D2A7_rep2; Mus musculus; RNA-Seq
RNA was isolated from 3 x 10e6 cells with the RNeasy Mini Kit (Qiagen, 74106) following the manufacturer's instructions with the following alterations. Cells were resuspended in 600 μl RLT buffer (with 2-Mercaptoethanol) and the homogenate further passed through a QiaShredder column (Qiagen, 79656) by centrifugation in a table top centrifuge for 2 minutes at full speed at room temperature (RT). The optional step after the second wash with RPE buffer was applied to dry the membrane. RNA was eluted with 85 μl H2O and supplied with 10 μl 10x DNAase buffer and 5 μl DNAse I (NEB, M0303S), mixed and incubated at RT for 20 min. After incubation RNA was purified by following the “RNA Cleanup” protocol in the RNeasy Mini Handbook. The optional step after the second wash with RPE buffer was applied to dry the membrane. RNA was eluted in 50 μl H2O and concentration determined with a NanoDrop 8000 Spectrophotometer. RNA-seq Library Preparation and Sequencing: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). Paired end 100 cycle sequencing was performed on a HiSeq 2500, HiSeq 4000, or NovaSeq 6000 System (all from Illumina) according to the manufacturer's instructions.