GSM8758826: MIA PaCa-2 cells, TNFα untreated, biological replicate 2; Homo sapiens; OTHER
During the last hour of TNF-α (0h, 24h) treatment, 0.5 mM 5-Ethynyl Uridine (5-EU) was added to the cells and incubated for 1 hour at 37ºC. Sub-cellular fractionation was done and RNA was isolated from nuclear fraction using TRIzol LS reagent (Ambion, Life Technologies) from TNF-ɑ treated (24h) and untreated (0h, control) MIAPaCa-2 cells. The nascent RNAs were captured using the Click-iT kit (Invitrogen) following manufacturer's protocol. In brief, Biotin Azide was covalently linked to EU-RNA using click chemistry and nacsent RNAs were pulled down by streptavidin Dyna beads and submitted for sequencing. The cDNA library was prepared using the KAPA RNA HyperPrep Kit [KR1351-v2.17] with RiboErase (HMR) library preparation kit following the manufacturer's protocol with a few modifications. In brief, for depletion of rRNA firstly streptavidin Dyna beads bound nascent RNA (SDBbnRNA) was hybridized with rRNA complementary DNA oligonucleotides followed by RNase H treatment. The rRNA depleted SDBbnRNA was captured using magnetic stand and the supernatant was collected and cleaned up using 2.2X KAPA Pure Beads followed by elution in DNase digestion master mix. This elute was then mixed with rRNA depleted SDBbnRNA which was separated earlier on magnetic separator device and proceeded for DNase treatment to remove rRNA and complementary DNA oligonucleotides duplex. DNase treated sample supernatant was then again collected and cleaned up using 2.2X KAPA Pure Beads as explained earlier and then eluted in diluted Fragment, Prime and Elute buffer and mixed with rRNA depleted, DNase treated SDBbnRNA. Henceforth, standard protocol was followed.