Illumina HiSeq 2500 sequencing; PHF3 binds RNA polymerase II via the SPOC domain and regulates transcription of neuronal genes - PRO-seq Timecourse
The protocol was adapted from Kwak et al, 2013. To isolate nuclei, cells were resuspended in cold buffer A (10 mM Tris-Cl pH 8, 300 mM sucrose, 3 mM CaCl2, 2 mM MgAc2, 0.1% TritonX-100, 0.5 mM DTT), incubated on ice for 5 min and transferred to a dounce homogenizer. After douncing 25 times with the loose pestle, cells were centrifuged at 700 g for 5 min. The pellet was resuspended again in buffer A, centrifuged and resuspended in cold buffer D (10 mM Tris-Cl pH 8, 25% glycerol, 5 mM MgAc2, 0.1 mM EDTA, 5 mM DTT), flash frozen with liquid nitrogen and stored at –80°C. For each run-on, 107 HEK293 nuclei was mixed with 106 of Drosophila S2 nuclei (10% for spike-in normalization) in 100 µL buffer D and incubated at 30°C for 3 min with 0.025 mM biotin-11-NTPs and run-on master mix (5 mM Tris-Cl pH 8, 2.5 mM MgCl2, 0.5 mM DTT, 150 mM KCl, 0.2 units/µL SUPERase In, 0.5% Sarkosyl).  Nascent RNA was isolated using TRIzol LS reagent according to the manufacturer's instructions, denatured at 65°C for 40 s, hydrolyzed using 0.2 M NaOH on ice for 20 min and neutralized with 1 volume of 1 M Tris-Cl pH 6.8. Buffer was exchanged with DEPC water using BioRad P-30 columns. Fragmented nascent RNA was subsequently enriched using Streptavidin M280 beads by rotating the samples for 20 min in binding buffer (10 mM Tris-Cl 7.4, 300 mM NaCl, 0.1% TritonX-100). Beads were subsequently washed twice with high-salt wash buffer (50 mM Tris-Cl pH 7.4, 2 M NaCl, 0.5% TritonX-100), twice with binding buffer and once with low-salt wash buffer (5 mM Tris-Cl pH 7.4, 0.1% TritonX-100). RNA was isolated from the beads using TRIzol reagent in two consecutive rounds and pooled together for ethanol precipitation. The RNA pellet was redissolved in DEPC H2O with 10 pmol of reverse 3' RNA adaptor and subjected to ligation using T4 RNA ligase I (NEB) at 16°C ON. RNA was isolated using Streptavidin M280 beads as previously described and 5' ends were repaired using Cap-Clip Acid Pyrophosphatase (Cellscript) and Polynucleotide Kinase (PNK, NEB) according to manufacturers' instructions. RNA was purified again with TRIzol and ethanol precipitation as before and subjected to 5' RNA adaptor ligation as for the 3' adaptor ligation. RNA was enriched by a third round of binding to Streptavidin M280 beads and TRIzol isolation.  RNA was retro-transcribed using RP1 Illumina primer and SuperScript III (Invitrogen) to generate cDNA libraries. Libraries were then amplified using KAPA HiFi Real-Time PCR Library Amplification Kit (Peqlab) and Illumina primers containing standard TruSeq barcodes and a random octamere sequence. Amplified libraries were subjected to electrophoresis on 2.5% low melting agarose gel and amplicons from 200 to 300 bp were excised, purified from the gel using NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel)