GSM8427604: C20 P23 D3; Homo sapiens; RNA-Seq
Total RNA was extracted from cells using Trizol, and RNA quantity and purity were determined by a NanoDropOne spectrophotometer (Nanodrop Technologies). RNA-seq library preparation was performed as described elsewhere and as follows (Link, Duttke et al. 2018). RNA isolation from C20 cells was carried out as described above and total RNA was enriched in poly-A tailed RNA transcripts by double incubation with Oligo d(T) Magnetic Beads (NEB, S1419S) and fragmented for 9 minutes at 94°C in 2X Superscript III first-strand buffer containing 10 mM DTT (Invitrogen, #P2325). The 10 μL of fragmented RNA was added to 0.5 μL of Random primers (Invitrogen, #48190011), 0.5 μL of Oligo d(T) primer (Invitrogen, #18418020), 0.5 μL of SUPERase inhibitor (Ambion, #AM2696), 1 μL of 10 mM dNTPs and incubated at 50°C for 3 minutes. Then, 5.8 μL of water, 1 μL of 10 mM DTT, 0.1 μL of 2 μg/μL Actinomycin D (Sigma, #A1410), 0.2 μL of 1% Tween-20 (Sigma) and 0.5 μL of SuperScript III (Invitrogen, #ThermoFisher 18080044) was added to the mix. Reverse-transcription (RT) reaction was performed at 25°C for 10 minutes followed by 50°C for 50 minutes. RT product was purified with RNAClean XP (Beckman Coulter, #A63987) and eluted in 10 μL in 0.01% Tween 20. The RNA-cDNA complex was then added to 1.5 μL of 10X Blue Buffer (Enzymatics, #B0110-L), 1.1 μL of dUTP mix (10 mM dATP, dCTP, dGTP and 20 mM dUTP), 0.2 μL of 5 μ/mL RNAseH (Enzymatics, #Y9220L), 1 μL of 10 U/μl DNA polymerase I (Enzymatics, #P7050L), 0.15 μL of 1% Tween 20 and 1.05 μL of nuclease free water; and incubated at 16°C for 2.5 hours or overnight. The resulting dsDNA product was purified using 28 μL of Sera-Mag SpeedBeads Magnetic Carboxylate (ThermoFisher, #65152105050250) diluted in 20% PEG8000, 2.5 M NaCl to a final 13% PEG concentration, washed twice with 80% EtOH, air dry and eluted in 40 μL of 0.05% Tween 20. The purified 40 μL of dsDNA was end-repaired by blunting followed by A-tailing and adapter ligation as described elsewhere (Heinz, Benner et al. 2010) using BIOO Barcodes (BIOO Scientific, #514104), 0.33 μL 1% Tween 20 and 0.5 μL T4 DNA ligase HC (Enzymatics, #L6030-HC-L). Libraries were amplified by PCR for 11–15 cycles using Solexa IGA and Solexa IGB primers (AATGATACGGCGACCACCGA and CAAGCAGAAGACGGCATACGA, respectively), and size selected for fragments (200-500 bp) by gel extraction (10% TBE gels, Life Technologies EC62752BOX). Eluted libraries were quantified using a Qubit dsDNA HS Assay Kit and sequenced on a NovaSeq 6000 (Illumina, San Diego, California).