title: GSM3891906: 7SK.no_PlaB.repl4.Total; Homo sapiens; RNA-Seq
library_construction_protocol: RNA was extracted using TRIZOL reagent (Thermo Fisher Scientific) following manufacturer instructions and quantified using a Nanodrop. For isolation of 4SU labelled RNA, 80 μg of RNA were used per replicate. The incorporated 4SU was biotinylated by incubating 1h 30 minutes at room temperature with EZ-link biotin-HPDP (Pierce) in biotinylation buffer (10mM Tris Ph 7.4, 1mM EDTA). Biotinylated RNA was then purified by phenol-chloroform extraction and loaded onto micro-MACS streptavidin columns (Miltenyi biotech). After 3 washes with warm (65°C) and 3 with room temperature washing buffer (100mM Tris pH 7.4, 10mM EDTA, 1M NaCl, 0.1% Tween 20), 4SU labelled RNA was then eluted directly into RLT buffer (Qiagen) by adding twice 100μl of 100mM DTT (Sigma Aldrich) and processed using the kit RNeasy MinElute (Qiagen), following manufacturer instructions. Libraries were prepared using the SMARTer Stranded Total RNA-Seq Kit, Pico Input Mammalian kit (Takara biotech), following manufacturer instructions. Four replicates per sample were sequenced on an Illumina HiSeq 4000 platform.