GSM8154027: Tether_HEK293T_Ox_3_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. Tether-seq libraries were generated following standard protocols using the SMARTer Stranded Total RNA-Seq Kit-Pico Input Mammalian, V2 (Takara Bio USA, cat. 634413).