GSM8730113: B16F10 shRNA Ctrl replicat2; Mus musculus; RNA-Seq
Cells were washed three times with cold PBS and lysed in RIP buffer (150 mmol/L KCl, 25 mmol/L Tris pH 7.4, 5 mmol/L EDTA,0.5 mmol/L DTT, 0.5%NP40) supplemented with protease inhibitor for 30min at 4°C. Lysates were cleared by centrifugation at 10.000G for 15min at 4°C. 1mg of lysate were mixed with anti-FLAG anti-A/G protein conjugated magnetic beads (MedeChemExpress, #HY-K0207, #HY-K0202 respectively) according manufacturer's instruction. Binding reaction was incubated on rotation overnight at 4°C. Beads were washed 4 times with RIP buffer and elution was completed with buffer elution A (0.15M glycine, pH3). After 10min, neutralization buffer (1M TrisHCl pH8.0) was added and followed by incubation with 10mg/mL of proteinase K (FischerScientific, #10103533) at 55°C for 1h. The RNA bound to FLAG-RIG was extracted using TRIzol protocol (Invitrogen) Library preparation was performed using 10 ng of total RNA following the Illumina Stranded Total RNA Ligation with Ribo-Zero Plus protocol  (Illumina). The final library profiles were assessed using the QIAxcel Advanced System (Qiagen), and concentrations were measured by qPCR using the SYBR Fast KAPA Kit.Indexed Libraries were pooled in equimolar ratios, and the pooled library was sequenced on the NovaSeq 6000 platform using an S2 flow cell with paired-end 150 bp reads, with 50 Mi fragments sequenced per sample.