title: GSM6368770: MEFs Casp8 and Mlkl knock-out with ectopic-reexpression of Casp8 rep 2; Mus musculus; RNA-Seq
library_construction_protocol: Total RNA was isolated from three independent cell cultures of Fadd-/-Mlkl-/- or Casp8-/-Mlkl-/- MEF reconstituted with empty vector (CTRL), FADD or Caspase-8 (Casp8), respectively. Briefly, RNA purification was performed by using RNeasy Kit (QIAGEN) according manufacturer instructions. RNA was quantified using the Quant-iT RiboGreen RNA assay (ThermoFisher) and quality checked by the 2100 Bioanalyzer RNA 6000 Nano assay (Agilent) or 4200 TapeStation High Sensitivity RNA ScreenTape assay (Agilent) prior to library generation. Libraries were prepared from total RNA with the TruSeq Stranded Total RNA Library Prep Kit according to the manufacturer's instructions (Illumina, PN 20020599). Libraries were analyzed for insert size distribution using the 2100 BioAnalyzer High Sensitivity kit (Agilent), 4200 TapeStation D1000 ScreenTape assay (Agilent), or 5300 Fragment Analyzer NGS fragment kit (Agilent). Libraries were quantified using the Quant-iT PicoGreen ds DNA assay (ThermoFisher) or by low pass sequencing with a MiSeq nano kit (Illumina). Paired-end 100 cycle sequencing was performed on a NovaSeq 6000 (Illumina). RNAseq data were processed using an automated pipeline maintained by St. Jude Children's Research Hospital. Briefly, reads were trimmed using Cutadapt within the Trim Galore wrapper and aligned to the mm10 genome with the M22 annotation (GENCODE) using STAR, and gene counts were subsequently calculated using RSEM. Gene counts were subsequently compared across conditions using the edgeR and limma and data were visualized using Glimma.