Stranded truseq rna seq protocol

To confirm the presence of read-through transcription, on the day of the ATAC/Omni-ATAC-seq experiment, total RNA was collected. Biological duplicates were carried out. For total RNA, cells were collected in 500 μl TRI reagent, and total RNA was isolated with Directzol-RNA Microprep Kit (Zymo Research #R1050) according to manufacturer’s instructions. The following steps were performed by the Core Unit Systemmedizin, Würzburg, Germany. For the total RNA libraries, both cytoplasmic and mitochondrial rRNA species were depleted. No rRNA depletion was performed for 4sU-RNA samples as rRNA only contributes about 40–50% of reads in 4sU-RNA samples. Library preparation for sequencing was performed using the stranded TruSeq RNA-seq protocol (Illumina, San Diego, USA). Libraries were sequenced on NextSeq 500 (Ilumina). 4sU-seq data for Fig. 1c, d and Supplementary Fig. 1d were taken from^{3 (link),5 (link)} and 4sU-seq data for Fig. 2e, f and Supplementary Fig. 7g–j from^{4 (link)}.

For both total and 4sU-RNA samples, library preparation for sequencing was performed using the stranded TruSeq RNA-seq protocol (Illumina, San Diego, USA) with 800 ng RNA as input for each sample. For the total RNA libraries, both cytoplasmic and mitochondrial rRNA species were depleted using the Ribo Zero Gold kit (Epicentre, WI, USA). No rRNA depletion was performed for 4sU-RNA samples as rRNA only contributes about 40–50% of reads in 4sU-RNA samples. In the PCR step, to enrich for fragment-ligated adaptors, only 13 cycles were used, which minimized PCR bias. Barcoded libraries were subjected to sequencing by synthesis at 2 × 101 nt on a HiSeq 2500 (Illumina). Quality monitoring and output settings were handled according to standards described by ‘t Hoen et al.54 (link).