GSM6260936: NET-seq ctrl rep1; Mus musculus; OTHER
Nascent RNA extraction was performed as described in Mayer and Churchman (2016). Around 10 million cells were collected per sample, and a 3% spike-in (by cell count) of Drosophila S2 cells was added per sample. Elongating Pol II-associated chromatin was collected by cellular fractionation, and RNA from the insoluble chromatin was extracted with Trizol for library preparation. NET-seq library preparation was performed as described in Mayer and Churchman (2016). Briefly, RNA was ligated with barcoded DNA linker and subjected to alkaline fragmentation. Fragmented RNA was size selected, purified, and reverse transcribed as first strand cDNA. cDNA was size selected, purified, and circularized. Circularized cDNA containing sequences of a subset of sequenced snRNAs, snoRNAs, and rRNAs were specifically depleted using biotinylated DNA oligos before being amplified with uniquely barcoded sequencing primers. Resulting NET-seq libraries were purified before sequencing.