GSM5320704: W303_sen1T1623E_R2; Saccharomyces cerevisiae W303; OTHER
Powders were defrozen and the resulting extracts were treated for one hour at 25°C with DNase I (165U/g of cells) to solubilize the chromatin and then clarified by centrifugating 20' at 20000g at 4°C.IgG and nickel purification steps were performed as previously described in the original protocol (granneman et al., 2009) with minor changes: i) IgG purification steps has been realized using M-280 tosylactivated dynabeads (15mg per samples) coupled with rabbit IgG. ii) To increase the stringency, washes following both IgG and nickel purification steps were done at high salt concentration (1 M NaCl). After over-night binding on nickel columns, sequencing adaptors were added on the RNA as described in the original procedure. Because we work on nascent transcripts, some changes were required to fit with our purpose: For instance, since nascent transcripts contain a 3' hydroxyl group that is protected inside the polymerase, we decided not to do the dephosphorylation step usually required because of the RNAse treatement that leads to 3' phosphate transcripts. Also, we modified both 5' and 3' adaptors in order to sequence RNA molecules from the 3' end so that we can determine the exact position of the polymerase. The 3' ligation has been realized with T4 rnl 2 truncated K227Q enzyme (NEB) instead of classical T4 RNA ligase. RNA-protein complexes were eluted in 400 µl of elution buffer (50mM Tris pH 7.8, 50mM NaCl, 150mM Imidazole, 0.1% NP-40, 5mM beta mercaptoethanol). Eluates were concentrated in Vivacon® ultrafiltration spin columns 30 KDa MWCO to a final volume of 120 µl. A protein fractionation step was performed using the Gel Elution Liquid Fraction Entrapment Electrophoresis (Gelfree) system from Expedeon. RNA pol II containing fractions were treated with 100 µg of proteinase K in a buffer containing 0.5% SDS. RNA was purified by two phenol chloroform isoamyl and one chloroform steps and precipitated in ethanol. RNA was reverse transcribed using the Reverse transcriptase IV (Invitrogen). The cDNA solution was used to perform multiple PCR reactions in a final volume of 25 µl using the following conditions: 0.4 µM of each primers, 0.2 mM dNTP, 2.5 U LA Taq DNA polymerase from Takara, 1X LA PCR Buffer II and 2 µl of cDNA per reaction with the programme: 95°C 2', 95°C 30'', 58°C 45'', 72°C 1', 72°C 5'. PCR were pooled and treated with 200 U of Exonuclease I (NEB) per ml of PCR reaction for 1 hour at 37°C. After Exonuclease I inactivation for 20' at 80°C, DNA was purified on PCR clean up columns (NucleoSpin® Gel and PCR Clean-up, Macherey-Nagel) and sent to sequence using Illumina technology (NextSeq sequencing).