GSM9263600: BPH1 ZC3H13 KO replicate 3; Homo sapiens; RNA-Seq
For RNA-seq and MeRIP experiments, total RNA was extracted from ZC3H13 control, ZC3H13 KO, ZC3H13-dCas9, and ZC3H13-overexpressing BPH-1 cells using QIAzol Lysis Reagent (Qiagen, 79306) according to the manufacturer's instructions. After isopropanol precipitation, DNAse I Turbo (ThermoFisher Scientific, AM2239) treatment was performed to remove any remaining DNA contamination.  For the MeRIP protocol, mRNA enrichment was performed using Dynabeads® mRNA Purification Kit (ThermoFisher Scientific, 61006) following the manufacturer's instructions. Three replicates per experimental condition were generated. 5 µg of mRNA was fragmented by adding 1 µl of 10X fragmentation reagent (Invitrogen, AM8740) before ethanol precipitation. The fragmented mRNA was recovered by centrifugation at 17,000 g for 30 min at 4ºC. The mRNA pellets were resuspended in DEPC water and 10% of the sample was used for inputs. Fragmented mRNA was diluted with 100 µl of 5X IP buffer (250 mM Tris pH 7.4, 500 mM NaCl, 0.25% NP-40) and incubated with 10 µg of anti-m6A antibody (Abcam, ab151230, RRID:AB_2753144) in the presence of RNase inhibitors, for 3 hours at 4ºC. 30 µl of SureBeads Protein A Magnetic beads (Bio-Rad, 1614013) were added to the mRNA-antibody mixture and incubated for 2 hours at 4ºC. The beads were washed three times with high salt IP buffer (50 mM Tris pH 7.4, 1 M NaCl, 1 mM EDTA, 1% NP-40), and twice with 1X IP buffer. To elute the immunoprecipitated mRNA, 200 µl of PK buffer (100 mM Tris-HCl pH 7.5, 50 mM NaCl, 10 mM EDTA) and Proteinase K (Invitrogen, AM 2548, Compound CID: 118855431) were added to the beads. To isolate RNAs, 1:1 Phenol:Chloroform was added and gently vortexed and kept at room temperature for 5 min. After centrifugation at 12,000 g, the aqueous phase containing the immunoprecipitated RNA was ethanol precipitated overnight. The immunoprecipitated RNA was resuspended in DEPC-treated water. RNA-seq library preparation was performed from three biological replicates for each condition following standard protocols. In brief, mRNA was enriched from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers followed by the second strand cDNA synthesis. The library was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification. The library was checked with Qubit (RRID:SCR_020553) and real-time PCR for quantification and bioanalyzer (RRID:SCR_019715) for size distribution detection. Quantified libraries were pooled and sequenced on Illumina platforms (RRID:SCR_016387).