GSM7730695: hNSCs 37C, RBM3, Proteintech; Homo sapiens; RIP-Seq
The hNSCs were treated with 4-SU at a final concentration of 100 μM, 24 hours prior to cells collection. Subsequently, the hNSCs cultured in a 6 cm plate for 14 days of differentiation were carefully washed twice with ice-cold PBS and subjected to cross-linkage using 4000 joules of UV irradiation for three times. Following this process, the cells were gently scraped off using 1 ml PBS and centrifuged at 300 g × 5 min, after which they were re-suspended in 300 μl of complete IP cell lysis buffer (20 μM of Tris-HCl pH 7.5, 10 μM of KCl, 5 μM of MgCl2, 0.5% NP-40, 1 μM of DTT (ThermoFisher, R0862), 40 units/ml of RNase inhibitor (Sangon Biotech, B600478), and EDTA-Free Protease Inhibitor Cocktail (Bimake, B14001)). Besides, after centrifugation, 100 ml of scraped cells were added with TRIzol (Vazyme, R401-01) labelled as “Input” for RNA analysis. The cells were then incubated on ice for 15 minutes and centrifuged at 12000 g for 15 minutes at 4C. After centrifugation, 30 ml of cell lysate supernatant were transferred to a new tube labelled as “Input” for protein analysis. The remaining supernatant was then treated with or without Rnase T1 (Thermo Scientific, EN0601), at a final concentration of 1 unit/ml, for 10 minutes at room temperature. Subsequently, the lysate supernatant was mixed with 10 ml of antibody-coupling agarose beads and incubated overnight at 4C with rotation. The next day, the samples were centrifuged at 3000 g for 5 minutes and the supernatant was carefully discarded. The samples were then washed thrice with 1 millilitre of ice-cold PBS. After the third centrifugation, the beads were resuspended with 200 microliters of IP buffer, and 20 microliters of the sample was taken for protein testing. Subsequently, protease K was added to a concentration of 100 micrograms/millilitre and incubated at 55 degrees Celsius for 2 hours. Finally, 1 millilitre of TRIzol was added to one sample to extract RNA for further sequence and qRT-PCR analysis. Bounded RNA was isolated from sample using RNeasy mini kit (Qiagen, Germany). Paired-end libraries were synthesized by using the TruSeq® RNA Sample Preparation Kit (Illumina, USA) following TruSeq® RNA Sample Preparation Guide. Briefly, The poly-A containing mRNA molecules were purified using poly-T oligo-attached magnetic beads. Purified libraries were quantified by Qubit® 2.0Fluorometer (Life Technologies, USA) and validated by Agilent 2100 bioanalyzer (Agilent Technologies, USA) to confirm the insert size and calculate the mole concentration. Cluster was generated by cBot with the library diluted to 10 pM and then were sequenced. The library construction and sequencing was performed at Shanghai Biotechnology Corporation.