title: GSM6032796: GF_48h_m4; Mus musculus; RNA-Seq
library_construction_protocol: Three PCLS slides per donor per assay condition have been pooled to ensure to get enough material for RNA Sequencing. After homogenization and lysis using in Qiazol buffer (Qiagen, Venlo, the   Netherlands), samples were mixed with 1/5th volume of chloroform (Sigma) and separated using phase extraction tubes. The RNA was then isolated with the RNeasy Lipid Tissue Mini Kit (Qiagen, Venlo, the  Netherlands) or the FavorPrep TM Tissue Total RNA Mini Kit (Favorgen, Vienna, Austria).  RNA samples were quantitatively and qualitatively assessed using the fluorescence-based Broad Range Quant-iT RNA Assay Kit (ThermoFisher) and the Standard Sensitivity RNA Analysis DNF-471 Kit on a 96-channel Fragment Analyzer (Agilent), respectively. Total RNA was normalized on the MicroLab STAR automated liquid platform (Hamilton). 100ng input was used for library construction with the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina #E7760, together with the QIAseq FastSelect RNA removal Kit (Qiagen) upstream and the NEBNext Multiplex Oligos for Illumina #E7600 downstream (all New England Biolabs). Fragmentation time was set to 15min. A total of 12 PCR cycles was used for the index PCR while the final libraries were eluted in 25 µL. Of note, the only deviation from the manufacturer's protocol was the use of Ampure XP beads (Beckman Coulter) at the double-stranded cDNA purification step, instead of the recommended SPRIselect Beads. Total RNA sequencing libraries were quantified by the fluorescence dye-based methodology High Sensitivity dsDNA Quanti-iT Assay Kit (ThermoFisher) on a Synergy HTX (BioTek). Library molarity averaged at 100.3 nM. Total RNA libraries were also assessed for size distribution and adapter dimer presence by the High Sensitivity NGS Fragment DNF-474 Kit on a 96-channel Fragment Analyzer (Agilent). Sequencing libraries were normalized on the MicroLab STAR (Hamilton), pooled and spiked in with PhiX Control v3 (Illumina). The library pool was subsequently clustered on an S2 Flow Cell and sequenced on a NovaSeq 6000 Sequencing System (Illumina) with dual index, paired-end reads at 2x100 bp length (Read parameters: Rd1: 101, Rd2: 8, Rd3: 8, Rd4: 101) reaching a target depth of approximately 60 million Pass-Filter reads per sample (6.5% CV, see).