Illumina HiSeq 2500 sequencing; PHF3 binds RNA polymerase II via the SPOC domain and regulates transcription of neuronal genes - RNAseq
RNA was collected from WT, PHF3-KO, and dSPOC HEK293 cell. Each biological condition was collected in three biological replicates. HEK293T cells were grown in Dulbecco's Modified Eagle's Medium (DMEM 4.5 g/l glucose) (Sigma) supplemented with 10% fetal bovine serum (Sigma), 1% L-glutamine (Sigma), 1% penicillin-streptomycin (Sigma) under 5% CO2 at 37°C.    To generate CRISPR/Cas9 PHF3 KO, gRNA targeting exon 3 was cloned between BbsI sites under the U6 promoter in the plasmid encoding Cas9-EGFP (PX45870). gRNA sequence for human PHF3 was 5'-TGATACTAGTACTTTTGGAC-3' and for mouse PHF3 5'-ATTCGGGTTCCTCGGCCGTC-3'. 48h after transfection with polyethylenimine (PEI; Polysciences), GFP-positive HEK293T cells were FACS-sorted and allowed to recover in culture for 4 to 7 days. Cells were subsequently FACS-sorted and GFP-negative cells were seeded 1 cell/well in 96-wells plates. After 14 to 20 days, surviving clones were expanded in culture, genomic DNA was isolated and PCR-amplified Cas9 target region was sequenced.  To generate CRISPR/Cas9 PHF3ΔSPOC, one gRNA target site on either side of the SPOC domain was selected in such a way that the PAMs are within the deleted fragment (5'-TGGCTCGATTGAACTTCATC-3' and 5'-GGTCCATCAAAAGGCACAAG-3'). A 150 bp ssDNA repair template was designed which introduces an XhoI restriction site at the junction of the two breaks without shifting the reading frame or altering the amino acid sequence (5'-TTAGGCTTTTTAATGTCATTTTCTAGTCCAGAGATGCCTGGAACTGTTGAAGTTGAGTCTACCTTTCTGGCTCGAGTGCCTTTTGATGGACCTGGTAGGTATACGTTTTAATAATAGGATAAGAATGAAATAACATGGGAGGTGGGACCA-3'). S-phase synchronized HEK293T cells were electroporated with 10 µg of purified Cas9, 12 µg of each in vitro transcribed gRNA and 4 µM repair template. Genomic DNA from the clones was PCR-amplified to check for the deletion of 4.5 kb and digested with XhoI to ensure that the repair template was used for homologous recombination-mediated repair.  8x10^6 HEK293 cells was mixed with 2x10^6 Drosophila S2 cells for spike-in normalization RNA was isolated from harvested cells using TRI reagent (Sigma) according to the manufacturer's instructions. RNA-seq libraries for HEK293T WT and PHF3 KO samples were prepared using SENSE Total RNA-Seq Library Prep Kit (Lexogen) according to the manufacturer's instructions.  Total isolated RNA was first treated with recombinant DNaseI (Roche), cleaned up using peqGOLD PhaseTrap A tubes (Peqlab), and rRNA-depleted using RiboZero kit (Epicentre)