GSM9198462: PRO-SEQ of shCtrl cells rep2; Homo sapiens; OTHER
PRO-SEQ experiments, Nuclear run-on RNA was extracted using TRIzol LS reagent PRO-SEQ: cells were washed twice with ice-cold PBS and then permeabilized by incubation in permeabilization buffer on ice for 10 minutes. Nuclei were subsequently washed twice with wash buffer and resuspended in 100 μl of freezing buffer. An equal volume of reaction buffer was added for the nuclear run-on reaction, which proceeded at 37°C for 7 min. Nuclear run-on RNA was extracted using TRIzol LS reagent (Invitrogen), following the manufacturer's instructions, and precipitated with ethanol. The recovered RNA was then ligated to a 3' adaptor using T4 RNA ligase. Biotinylated RNA was captured by incubation with streptavidin beads (Smart-Lifescience) for 20 minutes. While immobilized on the beads, the RNA underwent sequential enzymatic repairs: (1) 5' hydroxyl repair, (2) decapping, and (3) 5' adaptor ligation. Following these repairs, RNA was eluted from the beads using TRIzol reagent. The eluted RNA was then reverse-transcribed and amplified by PCR to generate sequencing libraries. The final cDNA library was purified, size-selected, and sequenced on an Illumina HiSeq2000 platform.