GSM6181193: mESC, STM2457, 6h, rep1; Mus musculus; RNA-Seq
For RNA extraction, cells were washed twice with ice cold 1x PBS and collected on ice. For total RNA isolation the RNeasy Plus Mini Kit (Qiagen) was used following the manufacturer's instructions. RNA-seq: All NGS library preps were performed with Illumina's Stranded mRNA Prep Ligation Kit following Stranded mRNA Prep Ligation Reference Guide (June 2020) (Document # 1000000124518 v00). For RNA-seq in HEK293T and C643 cells: Libraries were prepared with a starting amount of 350 ng for C643 samples and 1000 ng for HEK293T samples and amplified in 11 PCR cycles and 10 cycles respectively. For normalization, 2μl of a 1:300 dilution of ERCC spike-ins (Ambion) were added to the starting material for C643 samples and for HEK293T samples 2μl of a 1:100 dilution. Libraries were profiled in a DNA 1000 chip on a 2100 Bioanalyzer (Agilent technologies) and quantified using the Qubit dsDNA HS Assay Kit, in a Qubit 2.0 Fluorometer (Life technologies). All samples were pooled in equimolar ratio and sequenced on 1 NextSeq500 Highoutput FC, SR for 1x 80 cycles plus 10 cycles for the index read plus 1 dark cycle upfront Read 1. For RNAseq in male mESC cells upon 24 h of STM2457 treatment: Libraries were prepared with a starting amount of 1000 ng and amplified in 10 PCR cycles. For normalization, 2μl of a 1:100 dilution of ERCC spike-ins (Ambion) were added to the starting material. Libraries were profiled in a DNA 1000 chip on a 2100 Bioanalyzer (Agilent technologies) and quantified using the Qubit dsDNA HS Assay Kit, in a Qubit 2.0 Fluorometer (Life technologies). All samples were pooled in equimolar ratio and sequenced on 1 NextSeq500 Highoutput FC, SR for 1x 155 cycles plus 10 cycles for the index read and 1 dark cycle upfront Read 1. For RNAseq in RPE1 cells: Libraries were prepared with a starting amount of 1000 ng and amplified in 10 PCR cycles. For normalization, 2μl of a 1:100 dilution of ERCC spike-ins (Ambion) were added to the starting material. Libraries were profiled in a HS DNA chip on a 2100 Bioanalyzer (Agilent technologies) and quantified using the Qubit dsDNA HS Assay Kit, in a Qubit 2.0 Fluorometer (Life technologies). All samples were pooled in equimolar ratio and sequenced on 1 NextSeq500 Highoutput FC, SR for 1x 80 cycles plus 10 cycles for the index read and 1 dark cycle upfront Read 1. For RNAseq in male mESC upon STM2457 timecourse 3h-12 h: Libraries were prepared with a starting amount of 1000 ng and amplified in 10 cycles. For normalization, 2μl of a 1:100 dilution of ERCC spike-ins (Ambion) were added to the starting material. Libraries were profiled in a DNA 1000 chip on a 2100 Bioanalyzer (Agilent technologies) and quantified using the Qubit dsDNA HS Assay Kit, in a Qubit 2.0 Fluorometer (Life technologies). All samples were pooled in equimolar ratio and sequenced on 2 NextSeq500 Highoutput FC, SR for 1x 80 cycles plus 10 cycles for the index read plus 1 dark cycle upfront Read 1.  For RNAseq in female mESC single clones: Libraries were prepared with a starting amount of 500 ng and amplified in 10 PCR cycles. Two post PCR purification steps were performed to exclude residual primer and adapter dimers. Libraries were profiled in a DNA 1000 chip on a 2100 Bioanalyzer (Agilent technologies) and quantified using the Qubit dsDNA HS Assay Kit, in a Qubit 2.0 Fluorometer (Life technologies). All 12 samples were pooled in equimolar ratio and sequenced on 1 NextSeq500 Highoutput FC, SR for 1x 79 cycles plus 10 cycles for the index read and 2 dark cycles upfront Read 1.