title: GSM5191578: untreated replicate 7; Homo sapiens; RNA-Seq
library_construction_protocol: After treatment, cells were lysed in MagNA Pure LC RNA Buffer (Roche Diagnostics) and purified using the MagNA Pure 96 System (Roche Diagnostics). 10 ng of total RNA from each biological replicate were reverse transcribed to cDNA using Super Script IV Vilo (Thermo Fisher, 11766500, user guide MAN0015862). Libraries were generated with the AmpliSeq Library Plus Kit (Illumina Catalog number 20019103) according to the Reference Guide. Pipetting Steps for target amplification, primer digestion and adapter ligation were done with the mosquito automatic pipettor (ttp labtech) in a miniaturisation fashion. For the purifications before and after final library amplification, SPRI magnetic bead purification was chosen (Clean NGS, LABGENE Scientific SA) semi automated on multidrop (Thermo Fisher). Amplicon size and DNA concentration were measured using an Agilent High Sensitivity DNA Kit (Agilent Technologies, Waldbronn, Germany) according to the manufacturer's recommendation. Prior sequencing samples have to be normalized and pooled to 2nM final concentration on Biomek FXP workstation. Amplicon sequencing. Pooled libraries were sequenced on the Illumina NovaSeq 6000 Instrument, SBS (sequencing by synthesis) technology. 75 cycles end up with a minimum of 2 Mio SR per sample for analysing.