GSM5561650: y1-mouse-kidney-KOH; Mus musculus; OTHER
DNA was extracted using the MasterPure Complete DNA and RNA purification kit (MC85201, lucigen) preceded by adjusted approaches for homogenization and lysis of tissues. DNA from whole blood was extracted using the kit following manufacturer's instruction for the extraction from whole blood with RBC lysis. DNA from bone marrow originated from tibia and fibula. Bones were freed from any remaining tissue and broken at the knee joints. Placing this opening downward in a perforated 0.5 mL tube set in a 1.5 mL tube allowed to centrifuge out the bone marrow at 10,000 g for 20 s. 100 µL of RCB lysis buffer were added and mixed and then incubated at RT for 10 min while vortexing every 5 min. Samples were centrifuged at 10,000 g for 25 s and supernatant discarded. 300 µL of tissue and cell lysis buffer including proteinase K were added and incubated at 65 °C for 15 min while shaking at 1,500 rpm. Further extraction steps were performed following the kit's instructions. 2-70 mg of brain, heart, kidney, liver, lung, muscle or spleen tissue were added to a 1.5 mL tube containing 5-15 stainless steel homogenizing beads (0.9-2 mm, SSB14B, next advanced Inc.) and 100 µL tissue and cell lysis buffer including proteinase K. Samples were vortexed for about 20-30 min until tissue pieces were no longer visible. Beads were pelleted using a magnetic rack (DynaMag-2, Invitrogen) and the supernatant was transferred to a new tube containing 200 µL of tissue and cell lysis buffer including proteinase K. Samples were incubated at 65 °C for 15 min while shaking briefly every 5 min. After cooling the samples on ice for 5 min, further extraction steps were performed as instructed. Final DNA concentrations were determined using the Qubit dsDNA BR Assay Kit (molecular probes life technologies). 1 µg extracted DNA was mixed with 0.3 M KOH and incubated at 55°C for 2 hours. After ethanol precipitation, the pellet was dissolved in 20 µl elution buffer (Qiagen, 1014609). DNA was denatured by 3 min incubation at 85°C. In the phosphorylation step, 5'-OH end generated after KOH hydrolysis at the ribonucleotide site was phosphorylated using a mix of 10 U 3'-phosphatase minus T4 polynucleotide kinase (BioLabs, M0236L), 2.5 µl of 10X reaction buffer (BioLabs, B0201S), and 2.5 µl of 10 mM adenosine 5'-triphosphate (BioLabs, P0756S) in a total volume of 25 µl. After 30 min incubation at 37°C, the reaction was stopped by 20 min incubation at 65°C. The samples were bead-washed using CleanPCR kit (GC biotech, CPCR-0050). In the ligation step, ARC140 adaptor was ligated to the 5'-OP. Denatured DNA was ligated using a mix of 10 U T4 RNA Ligase 1 (ssRNA Ligase) (BioLabs, M0204S), 5 µl of 10X reaction buffer (BioLabs, B0216S), 0.5 µl of 2 mM adenosine 5'-triphosphate (BioLabs, P0756S), 25 µl PEG8000(50%), 5 µl of 10 mM [Co(NH3)6]Cl3, and 0.5 µl of 100 µM oligo ARC140 in a total volume of 50 µl. After overnight incubation at the room temperature, the samples were twice beads-washed and dissolved in 14 µl elution buffer. In second strand synthesis step, denatured DNA was polymerized at the room temperature for 5 min using a mix of 4 U T7 DNA polymerase (unmodified) (BioLabs, M0274L), 2 µl of 10X reaction buffer (BioLabs, B0274A), 2 µl of 2 mM adenosine 5'-triphosphate (BioLabs, P0756S), 0.8 µl of BSA (1 mg/ml) (BioLabs, B9000S), and a heterodimer oligo, ARC76/77 in a total volume of 20 µl. After beads-wash and elution, the libraries were amplified by PCR reactions using KAPA HiFi hotstart readymix (2X) kit (KAPA biosystems, KK2602, 07958935001) with PCR parameters as follows: 95 °C for 45 s, followed by 18 cycles at 95 °C for 15 s, 65 °C for 30 s, and 72 °C for 30 s. The amplified libraries were beads-washed and eluted in 20 µl TE. The library concentration was measured using Qubit® dsDNA HS Assay Kit (Life technologies, molecular probes, Q32854). The library quality was checked by the Agilent 2100 bioanalyzer system (Agilent Technologies). Illumina NextSeq 500 system was used for paired-end sequencing.