GSM5293216: dsRNA-seq_WT_Input_2; Homo sapiens; OTHER
NSUN2 knockout and wild-type A549 cells were prepared as described (3 replicates each, four 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) 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. Input RNA and J2-bound dsRNA were extracted using TRIzol, with small RNA (< 200 nt) removal by RNA Clean & Concentrator (Zymo Research). The "purified dsRNA" and "input" was subjected to library construction with SMARTer® Stranded Total RNA-Seq Kit v2. RNA-Seq. All libraries were sequenced on Illumina NovaSeq 6000 with paired-end 50 bp read length.