GSM8751519: AS-G3BP-2; Homo sapiens; OTHER
For sodium arsenite treatment, HeLa cells with 80% confluence were cultured in prewarmed medium containing 0.5 mM of sodium arsenite (Sigma-Aldrich) under culture conditions for 1 h. The cells that only changed the medium simultaneously were set as the control group. Fixed cells by 4% PFA for 15 min. After washed with PBS and added permeabilization buffer  and incubated on ice for 15 min. Washed the cells and blocked with blocking buffer at room temperature for 1h. Diluted the antibody of G3BP1 (Abcam, ab181150; 1:200 dilution) in antibody buffer. The concentration of the IgG antibody (Abclonal, AC005) should be the same as the primary antibody. Next, cells were incubated with antibodies on the rotation shaker at 25°C for 2 h. Washed cells with primary antibody wash buffer and PBST (PBS with 0.1% Tween-20) for three times respectively.The guinea pig anti-rabbit IgG (heavy & light chain) antibody (Antibodies Online, ABIN101961) was labeled with streptavidin through the Streptavidin Conjugation Kit (Abcam, ab102921). Subsequently, added 12.5 μM biotinylated T7-N9 oligo-M-EB (1:100 dilution; sequence 5'Bio-TCACTGTAGTTGTACAGACTCCATAATACGACTCACTATAGGGATAATGAGCAGACGTGTGCTCTTCCGATCT-NNNNNNNNN-3') and the streptavidin-secondary antibody (1:150 dilution) into the low adhesion tube and incubated at 4°C for 20 min on the rotation shaker to assemble the streptavidin-antibody-biotinylated-oligo triplex. Purified the triplex by the Amicon® Ultra Centrifugal Filter 100 kDa MWCO (Millipore) to remove the excessive biotinylated oligo. Dissolved the triplex with primary antibody wash buffer supplemented with 3% BSA and incubate the cells on the shaker at 25 °C for 45-60 min. Washed the cells with primary antibody wash buffer, wash buffer I, wash buffer II and PBST three times, respectively. Then rinsed the well with PBS twice times, incubated with PBS at 37°C for 10 min, ramped to 25°C for 5-10 min and hold at 25°C. The in situ reverse transcription was performed by SuperScript™ III (Thermo Fisher Scientific) at 37°C for 1 h. Washed the cells with 10 mM Tris HCl (pH 7.5), performed second strand synthesis by DNA Polymerase I (E. coli) (NEB), E. coli DNA Ligase (NEB) and RNase H (NEB). Added T4 DNA polymerase (NEB) and incubate at 16°C for 15 min for end repair. Then cells were digested with Proteinase K solution (Thermo Fisher Scientific) at 55°C for 30 min. Purified the dsDNA product by DNA Clean & Concentrator-5 Kit (Zymo research). Next, transcript and linear amplificated with T7 RNA Polymerase (Vazyme) at 37°C for 2-4 h and purified RNA by Dynabeads MyOne Silane (Thermo Fisher Scientific). Assembled the T4 Polynucleotide Kinase (NEB) reaction and incubated at 37°C for 30 min. After silane beads clean-up, performed the 3' adapter (sequence 5'Phos-AGATCGGAAGAGCGTCGTG-3'Bio) ligation by T4 RNA Ligase 2 truncated KQ (NEB) at 25°C for 4 h or 16°C overnight. Then purified the RNA with silane beads. Reverse transcription (primer sequence 5'-ACACGACGCTCTTCCGATCT-3') was performed by M-MLV Reverse Transcriptase (Promega). Purified with silane beads and library amplification by NEBNext® Ultra™ II Q5® Master Mix (NEB). The library was purified by AMPure XP beads (Beckman) and 2% low melting agarose (Solarbio) gel with size selection between 200 and 500 bp. Purification cDNA from agarose gels through Gel DNA Recovery Kit (Zymo research) and sequencing.