GSM9376450: END_seq_siREXO4_RNaseH1_Rep1; Homo sapiens; OTHER
A total of 5 million HeLa cells were collected by trypsin and washed with PBS. The cells were embedded agarose plug (Bio-Rad) to minimize externally produced DNA damage. Three plugs were produced for every cellular condition for each experiment. Plugs were incubated with proteinase K solution (TransGen) at 50 ºC for 1 hr and then at 37 ºC overnight, followed by consecutive washes in wash buffer (10 mM Tris (pH 8.0) and 50 mM EDTA) and then in a TE Buffer (10 mM Tris (pH 8.0) and 1 mM EDTA). Plugs were incubated with RNase A solution (TransGen) at 37 ºC for 1 hr and washed three times with wash buffer. The plugs were blunted with Exonuclease VII (NEB) and Exonuclease T (NEB), respectively. After washing, two consecutive enzymatic reactions were performed on the plugs: A-tailing with Klenow fragment (3'→5' exo-) (NEB), followed by ligation to biotinylated END-seq adaptor 1. The plugs were washed between each indicated step. Next, DNA was recovered by first melting the agarose plugs and then digesting the agarose with -agarase I (NEB) at 43 ºC for 45 min, and dialyzed using dialysis membrane (Millipore). The sample was incubated with proteinase K (TransGen) at 50 ºC for 15 min and sheared to a length between 200 and 300 bp by Bioruptor sonicator (Diagenode). Following ethanol precipitation, the biotinylated DNA fragments were purified using streptavidin magnetic beads (Invitrogen). After end-repair with a combination of T4 DNA polymerase (NEB), DNA Polymerase I, Large (Klenow) Fragment (NEB), and T4 polynucleotide kinase (NEB), the fragments were A-tailed using Klenow fragment (3'→5' exo-) (NEB). Subsequently, END-seq adaptor 2 was ligated using the NEB Quick Ligation Kit (NEB). Libraries were prepared by first digesting the hairpins on both adapters with USER enzyme (NEB) and PCR-amplifying of the supernatant and bead-bound DNA mixtures for 16 cycles using TruSeq index adapters (Vazyme). Finally, the libraries were purified with VAHTS DNA Clean Beads (Vazyme) followed by agarose gel extraction, and then sequenced on a DNBSEQ-T7 for 150-bp paired-end reads.