GSM4979826: XRN-1(+) rep3; Homo sapiens; OTHER
Human genomic DNA was isolated from the K562 cell line and fragmented with DNase. After heating at 75°C for 10 min, digested DNA was purified and the desired size range of 200–1000 bp was confirmed by agarose gel electrophoresis. The DNA was then incubated with T4 DNA Polymerase in presence of dNTPs to repair the ends. The fragmented genomic DNA was then ligated to the two annealed adapters using T4 DNA ligase. All of the purified DNA was then used for PCR amplification. The amplified DNA was then used as a template for IVT by T7 RNA polymerase. The IVT products were denatured and renatured. The cleavage reaction was performed by incubating the renatured IVT products in 2 mM MgCl2, 100 mM KCl and 30 mM Tris-HCl pH 7.0 for 1 h at 37°C. One microgram of RNA (either treated with RppH and XRN-1 enzymes or the untreated control) was reversed transcribed. TdT and dCTP were added to the denatured cDNA and incubated at 37°C for 30 min. Then ddCTP was added to the reaction mixture and incubated at 37°C for additional 30 minutes to block free 3′-OH ends, followed by incubation at 70°C for 10 minutes to inactivate TdT. For s168-P sample, an RNA oligo containing a monophosphate moiety on both 5′ and 3′ ends was ligated to the self-cleavage products of the 168 nt fragment of hovlinc ribozyme in the presence of the RtcB ligase. Libraries were prepared for sequencing using standard Illumina protocols