NextSeq 550 sequencing; PHF3 binds RNA polymerase II via the SPOC domain and regulates transcription of neuronal genes - Targeted SLAM-seq
SLAM-seq was performed as described (Herzog et al., 2017). HEK293T cells seeded into 6 cm dishes the day before the experiment were incubated in standard culture medium containing 100 µM 4-thiouridine (s4U; ChemGenes) for 12 h with media exchanges every 3 hrs. Subsequently, cells were washed twice with PBS and incubated with 10 mM uridine-containing medium. Cells were harvested using TRI Reagent (Sigma) at timepoints 0 h, 6 h and 12 h after removal of s4U.  RNA was isolated according to the manufacturer's instructions including 0.1 mM DTT during isopropanol precipitation and resuspended in 1 mM DTT. Isolated RNA was treated with 10 mM iodacetamide to alkylate s4U and subjected to ethanol precipitation. Alkylated RNA was resuspended in water and treated with TURBO DNA-free Kit (Invitrogen) according to the manufacturer's instructions.  RNA was reverse transcribed using oligo(dT) primer. Subsequently, RNA was removed using RNAse H (NEB) according to the manufacturer's instructions and cDNA was amplified using INA-specific forward primer (5'- CACGACGCTCTTCCGATCTNNNNNNTCTGTCCAGCAGTCACTTCG-3') and oligo(dT) primer (5'-GTTCAGACGTGTGCTCTTCCGATCT-(T)n-V-3') for 20-25 cycles. Library amplification was performed using Illumina FWD (5'- AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3') and REV (5'- CAAGCAGAAGACGGCATACGAGATNNNNNNGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3') index primers.