GSM5288655: HepG2_dsRNA_rescue_ALKBH7-mutant_rep2_fwd; Homo sapiens; OTHER
Basically, as the first step of harvesting cells, the cells were washed once with DPBS buffer for each plate after aspirating the media. ALKBH7 knockdown HepG2 cells were prepared as described (two replicates each, five 15-cm plates per replicate). For each sample, 250 µL of Protein G Dynabeads (Invitrogen™, 10004D) was washed twice and resuspended in 1 mL of IP wash buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM MgCl2, 0.5% NP-40). 25 µg of anti-dsRNA mAb (J2)28,30 was added to the beads and incubated for 1 hour at room temperature, followed by another 1-2 hours at 4 °C. Cultured cells were washed once with cold DPBS. Each plate of cells was soaked in 5 mL cold DPBS and collected by cell lifters, combining 5 plates for each sample. The cell suspension was spun at 300 g at 4 °C for 2 mins. Cells were resuspended in 1 mL NP-40 lysis buffer (10mL solution: 50 mM HEPES pH 7.5, 150 mM NaCl, 2 mM EDTA, 10 mM MgCl2, 0.5% NP-40, plus 50 µL TURBO™ DNase (2 U/µL, AM2238, Invitrogen), 500 µL SUPERase•In™ RNase Inhibitor (20 U/µL, AM2694, Invitrogen), Pierce™ EDTA-free Protease Inhibitor Tablet) and incubated at 4 °C for 30 mins. The lysate was spun at 16,000 g at 4 °C for 5 mins. The supernatant was transferred to a new Eppendorf tube and 10 µL of that was kept as the “input”. The lysate was diluted to 4.75 mL with IP wash buffer. 250 µL of J2-Dynabeads was added to the lysate and incubated at 4 °C for 2 hours. The magnetic beads were washed once with 1 mL IP wash buffer and transferred to a new Eppendorf tube. The beads were further washed twice by 1 mL of IP wash buffer. Then the enriched dsRNA was eluted from beads and purified by TRIzol reagent. Around 50 ng human cellular double-stranded RNA by J2-IP (dsNRA) was fragmented into 40-70 nt using RNA Fragmentation Reagent (AM8740, Invitrogen) following the manufacturer's protocol and Oligo Clean & Concentrator (Zymo Research). These RNAs can be from diverse cellular treatments such as siALKBH7+empty-vector vs. siALKBH7+wild-type ALKBH7 vs. siALKBH7+mutant ALKBH7. Then the library construction procedures will be conducted with T4 PNK end repair, RNA 3'-adapter ligation, HIV RT reverse transcription, cDNA 3'-adapter ligation and PCR amplification respectively, according to standard steps in section “DAMM-seq for methylated site detection”. All libraries were sequenced on Illumina NovaSeq 6000 with paired-end 50 bp read length.