Name: GSM6603483
Instrument: NextSeq 500
Strategy: OTHER
Source: TRANSCRIPTOMIC
Selection: other
Layout: SINGLE
Construction protocol: PRO-seq datasets were prepared as described in Mahat 2016 (https://doi.org/10.1038/nprot.2016.086). Briefly, between 8 to 12 million nuclei per dataset were used for the PRO-seq transcription run-on using a mixture of rNTP and Biotin-11-CTP (PerkinElmer NEL542001EA). Total RNA was extracted using a phenol/chloroform precipitation. Isolated RNA was fragmented using base hydrolysis with NaOH. Biotinylated fragmented nascent transcripts were isolated using a first streptavidin Dynabeads M-280 (Invitrogen 11206D) pull down, and the VRA3 RNA adaptor was ligated at their 3' end. A second streptavidin bead pull down was performed, followed by the enzymatic modifications of the RNA fragment 5' ends with a pyrophosphohydrolase and a polynucleotide kinase, and the VRA5 RNA adaptor was ligated at their fixed 5' ends. A third streptavidin bead pull down was performed, followed by the reverse transcription of the resulting adaptor-ligated libraries. The libraries were cleaned up with AMPure XP beads (Beckman Coulter A63881). Then, the libraries were amplified using 13 PCR cycles, and cleaned up again with another round of AMPure XP beads. The resulting library concentrations were measured with the Qubit dsDNA high sensitivity assay (Invitrogen Q32851), and their size distributions assessed using the Agilent High Sensitivity D1000 ScreenTape. PRO-seq datasets were sequenced using an Illumina NextSeq as single-end 75 bp long reads.