GSM8154057: SHAPE_COX1_P1_no_1M7_A; Homo sapiens; RNA-Seq
RNA was harvested under non-denaturing conditions using a hypotonic lysis buffer (HLB). The pelleted cells were resuspended in HLB (20 mM HEPES, pH 8, 2 mM MgCl2, 0.2 mM EGTA, 10% glycerol, fresh 1 mM DTT) and freeze-thawed 3x and then placed on ice. After adding NaCl to 400 mM, samples were iced for 5 min and then centrifuged for 15 min at 4°C.The supernatant was collected, treated with proteinase K and 1% SDS and incubated at 37°C for 45 min. RNA was extracted 2x with phenol:chloroform:isoamyl (24:24:1) and 2x with chloroform. The aqueous layer was treated with Turbo DNAse and then buffer exchanged into folding buffer. RNA was reverse transcribed by first annealing to the RT primer (2 pmol) by heating at 65°C for 5 minutes and then cooling on ice. The RT samples (20 μL) were then prepared by adding 0.4 volumes of freshly prepared 2.5x MaP buffer (125 mM Tris-HCl, pH 8, 187.5 mM KCl, 15 mM MnCl2, 25 mM DTT, 1.25 mM dNTPs) and 1 μL of SuperScript II and incubating at 42°C for 3 hours. The enzyme was heat-inactivated by heating at 70°C for 15 minutes and the cDNA product was purified with 3 volumes of SPRI beads. The RT cDNA was amplified using Phusion polymerase and 10 pmols of PCR primers and purified with 3 volumes of SPRI beads. The PCR product was further amplified using 10 pmols of primers containing sequencing indexes and isolated using 1.5 volumes of SPRI beads.