GSM2218708: SKMM1 cells expressing FAM46CD90A,D92AGFP for 72 hrs rep2_polyA RNA; Homo sapiens; RNA-Seq
total RNA was extracted and poly(A)+ RNA was fractionated in terms of length. Fraction 5 and 6 represents transcripts with longest poly(A) tails. RNA isolation was performed with Trizol reagent (Invitrogen, 15596) according to the manufacturer’s instructions. Poly(A) fractionation was performed as previously described with the following modifications [Meijer, H.A. et al. A novel method for poly(A) fractionation reveals a large population of mRNAs with a short poly(A) tail in mammalian cells. Nucleic Acids Res 35, e132 (2007)]. In brief, 80 µg of total RNA was mixed with 400 µl GTC buffer (4 M guanidine thiocyanate, 25 mM sodium citrate, pH 7.1, 2% β-mercaptoethanol) and 750 pmoles of biotinylated 5’biotin –TEG-oligo(dT)25 (Future Synthesis). 850 µl of “dilution buffer” (6× SSC, 10 mM Tris–HCl, pH 7.4, 1 mM EDTA, 0.25% SDS, 1% β-mercaptoethanol) was added, the mixture was incubated at 70°C for 5 minutes, and then it was centrifuged at 12 000 rpm for 10 min at RT. The resulting supernatant was mixed with 100 µl of M280 beads (Invitrogen; 60210) washed 3 times with 0.5x SSC buffer. After binding, samples were washed 3 times with 0.5x SSC buffer for 10 minutes. RNA fractions were eluted with decreasing concentrations of SSC buffer (from 0.2x SSC to 0.025x SSC) and the final elution was performed with water. RNA was precipitated and subjected to further analysis. 1 µg of total RNA was treated with Turbo-DNase (Life Technologies) according to the manufacturer’s instructions. Then, rRNA was removed from samples using a Ribo-Zero Kit (Epicentre), according to the manufacturer’s protocol, and samples were spiked-in with external RNA (ERCC RNA Spike-In Mix; Life Technologies). For libraries prepared from poly(A)+ fractions, the ribo-depletion step was omitted. Finally, RNA libraries were prepared using a TruSeq RNA Sample Preparation Kit (Illumina) or a KAPA Stranded RNA-Seq Library Preparation Kit (Kapa Biosystems) according to the manufacturer’s instructions. Quality of libraries was determined by chip electrophoresis performed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Inc.).
GEO Accession
GSM2218708