GSM4460022: HepG2_dsRNA_siALKBH7-rep1; Homo sapiens; OTHER
Twenty 15-cm plates of HepG2 cells were prepared for 48-hour knockdown (siControl, siALKBH7, 5 plates per replicate) or twenty 15-cm plates of HeLa cells were prepared for 24-hour overexpression (o/e Control, o/e ALKBH7, 5 plates per replicate) or twenty 15-cm plates of HepG2 cells were prepared for wild-type vs. ALKBH7 knockout (WT, ALKBH7 KO, 5 plates per replicate). At first, for every 5 plates of cells, 250 uL Protein G Dynabeads were washed twice and then resuspended in 1 mL 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) was incubated with such 250 µL of beads for 1 hour at room temperature and then moved to 4 °C for another 1-2 hours. During this incubation, for every 5 plates, the cell culture media was removed, and cells were washed by cold DPBS. Then cells were collected by cell lifter in 5 mL cold DPBS and merged into one 50 mL centrifuge tube. The cell suspension was spun at 300g at 4 °C for 2 min and the cell pellet was resuspended with 1 mL NP-40 lysis buffer prepared in 10 mL volume: 50 mM HEPES pH 7.5, 150 mM NaCl, 2 mM EDTA, 10 mM MgCl2, 0.5% NP-40, plus 50 uL TURBO™ DNase (2 U/µL, AM2238, Invitrogen) and 500 uL SUPERase•In™ RNase Inhibitor (20 U/μL, AM2694, Invitrogen). Incubate the cell suspension at 4 °C for 30 minutes, followed by a spin at 16,000g at 4 °C for 5 min. The supernatant was transferred to a new Eppendorf tube and keep 10 uL of that as input with TRIzol added. The 1 mL lysate was dilute to 5 mL with IP wash buffer and then 250 uL of J2-Dynabeads was added for an incubation at 4 °C for 2 hours. The magnetic beads were washed once with 1 ml of IP wash buffer and transferred into a new Eppendorf tube by 500 µL of IP wash buffer. The beads were further washed twice by 1 mL of IP wash buffer, and J2-bound dsRNA was extracted with TRIzol reagent. Around 50 ng human cellular double-stranded RNA by J2-IP (dsNRA) was fragmented into 40-50 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 vs. siControl or o/e Control vs. o/e ALKBH7 or wild-type vs. ALKBH7 KO. 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 “DAOMM-seq for methylated site detection”. All libraries were sequenced on Illumina NovaSeq 6000 with single-end 100 bp read length.
GEO Accession
GSM4460022