GSM8413685: n3_Input; Mus musculus; RIP-Seq
Co-Immunoprecipitation (Co-IP) was performed as previously described . In brief, P0 whole brain tissue was lysed in lysis buffer (300mM NaCl, 20mM Tris, 1mM EDTA, 0.5% Nonidet-40 (NP-40), pH 7.4) supplemented with protease inhibitor (Sigma-Aldrich/Merck, Germany) and incubated for 30min on ice, triturating every 10min 20 times. After a 10min centrifugation at 13000rpm, the supernatant was collected. The samples were diluted dropwise to reach a salt concentration of 100 mM and NP-40 concentration of 0.15% using equilibration buffer (20mM Tris, 1mM EDTA, pH 7.4). Protein concentrations were determined with Bradford reagent (Bio-Rad, United Kingdom). 10% input was saved and equal amounts of protein were used for all Co-IPs. Protein G Dynabeads (10004D, Thermo Scientific, USA) were washed with Co-IP buffer (100mM NaCl, 20mM Tris, 1mM EDTA, 0.15% NP-40 pH 7.4), and incubated with Co-IP antibodies at 4°C overnight. The following antibodies were used: anti-FOXG1 (#61211, Active Motif, USA), IgG (#C15410206, Diagenode, Belgium). Antibody-coupled beads were washed once with Co-IP buffer before Co-IP. Cell lysates were precleared with Protein G Dynabeads for 1h at 4°C, subsequently transferred to antibody-coupled beads, and incubated overnight at 4°C in rotation. In FOXG1-coIP_RNAse samples, RNAse A (1:400, R4642, Sigma-Aldrich/Merck) was added to this overnight step. Antibody-coupled beads were washed 3 times with Co-IP buffer. The washed beads were resuspended in RLT buffer of the RNA isolation kit and purified according the manufacturer's instructions (RNAeasy Mini Kit, Qiagen, Germany). Total RNA extract of FOXG1- and IgG-coIP respectively were used for RIPseq. The cDNA libraries were constructed using TruSeq total RNA sample preparation kit (Illumina, USA), following the manufacturer's instructions and sequenced on Illumina NovaSeq as paired-end 101bp reads.