GSM7873767: RIP2; Homo sapiens; RIP-Seq
10 million pancreatic cancer MiaPaCa cells were cultured in 15cm plastic culture dish (nunc) in RPMI1640 (Nacalai Tesque, Kyoto, Japan) with 10 % FBS under 5% CO2 at 37℃ condition.  V1SRP9V1-3xFLAG and SRP9V2-3xFLAG gene were prepared by DNA synthesis (Eurofins), of which sequences were confirmed by Sanger sequencer (ABI). pIRES2-acGFP1-NLS-SRP9V1-3xFLAG vector and pIRES2-acGFP1-NLS-SRP9V2-3xFLAG vector were designed to express exogeneous SRP9V1-3xFLAG or SRP9V2-3xFLAG genes in the split protein by the internal ribosome entry site signal (IRES, an RNA element that enables cap-independent initiation of translation as part of the process of protein synthesis) from acGFP1 under NLS, which is fused with exogeneous SRP9V1-3xFLAG or SRP9V2-3xFLAG. The SRP9V1 or SRP9V2 peptide is translated in frame with 3xFLAG peptide (MDYKDHDGDYKDHDIDYKDDDDK), respectively. Transfection of pIRES2-acGFP1-NSL-SRP9V1 vector or pIRES2-acGFP1-NSL-SRP9V2 vector plasmid performed by electroporation with Nepagene system (Nepagene), which can allow the preservation of membrane damages during transfection, with compared to lipofectamine methods (Takara). The cells were grown in the RPMI1640 with 10% FBS and 200mg/ml Geneticin (Nacalai) for two weeks to select gene transfected cells. The expression of protein was confirmed by acGFP signal under observation with fluorescent All-In-One microscope (Keyence). The 5x106 of acGFP expressing cells were collected by fluorescence-activated cell sorting (FACS; BD) and cultured in RPMI1640 with 10% FBS for 24 hours and subjected to protein extraction. For protein extraction, adherent cells were washed with PBS(-) (Nacalai) for three times in 15cm plastic culture dish, to exclude amino acids in the medium, which can interfere potentially with cross-linker. Cells were harvested with plastic cell-scrapers, and collected in 2ml tubes. Samples were centrifuged at 1200 rpm at 4oC for 5 min. Collected cell pellets were lysed in the 1ml HEPES-RIPA (150 mM KCl, 50 mM HEPES-NaOH (pH7.4), 1% NP-40, and 1xRoche cOmplete, proteinase inhibitor, prepared from 50x stock reagent (catalog number 04693116001; Roche. Samples were centrifuged at 1200 rpm at 4℃ for 5 min. Collected cell lysates were incubated for 30 min at room temperature with crosslinking agent, BS3 (bis(sulfosuccinimidyl)suberate) (Thermo Fisher Scientific), which do not permeate cell membranes, so we permeabilized with the treatment of a surfactant, NP-40, as mentioned above. We used a newly purchased BS3 (Lot. YB351210), considering it absorbs moisture from the atmosphere and becomes inactive. After the cross-linking reaction, 1/50 (final concentration 20 mM) of 1 M Tris-HCl, pH 7.5 was added to quench the excess cross-linking agent and gently mix by inverting for 15 minutes. For purification of target SRP9V1-3xFLAG or SRP9V2-3xFLAG proteins, samples were subjected to the immunoprecipitation. To this end, 20ul of anti-DDDDK antibody, which binds to FLAG tag sequence was added to each sample, and incubated at 4℃ for overnight (16 hr), and then purified with protein A and G conjugated magnetic beads ( Bio-Rad). The immunoprecipitated SRP9V1-3xFLAG or SRP9V2-3xFLAG protein, which crosslinked with endogenous RNA was wash with 1ml HEPES-RIPA (150 mM KCl, 50 mM HEPES-NaOH (pH7.4), 1% NP-40）; then, 1ml high salt HEPES-RIPA (300 mM KCl, 50 mM HEPES-NaOH (pH7.4), 1% NP-40); and once again 1ml HEPES-RIPA (150 mM KCl, 50 mM HEPES-NaOH (pH7.4), 1% NP-40, which were to exclude non-specific bound substances to protein A and G conjugated magnetic beads. Finally, samples were treated with 100 mg of 3xDYKDDDDK peptide (FP-1; Sigma), to elute endogenous RNA bound SRP9V1-3xFLAG or SRP9V2-3xFLAG proteins (hereinafter referred to as "RIP1" and "RIP2" respectively). RNA was extracted from samples by RNeasy extraction kit (Qiagen). 100 ng of RNAs were subjected to high-speed sequencing system of NovaSeq6000 (Illumina). As controls, input RNAs were prepared and used from geneticin-selected transfected cells with SRP9V1-3xFLAG or SRP9V2-3xFLAG (hereinafter referred to as "INPUT1" and "INPUT2" respectively). Each Illumina sequencing library was prepared using SMARTer Stranded Total RNA-seq Kit v2 -Pico Input Mammalian (Takara Bio).