GSM6605710
Pellets from 100 ml culture infected with either T4 phage WT, T4 phage ModB R73A,G74A (R73A) or the uninfected control (LB) were resuspended in 2 ml Ni-NTA wash buffer (10 mM imidazole, 50 mM Tris-HCl pH 7.5, 1 M NaCl, 1 M urea, 5 mM MgSO4, 5 mM β-mercaptoethanol, 5 % glycerol, pH 8.0, EDTA-free protease inhibitor) on ice and lysed by sonication (6 min, 50 % amplitude, 0.5 s pulse). The lysate was cleared from cell debris by centrifugation at 21,000 x g, 4 °C, 30 min. 1.9 ml supernatant, 50 µl Ni-NTA agarose beads (Jena Bioscience, equilibrated in Ni-NTA wash buffer), 80 U murine RNase inhibitor and 4.72 µg of rS1 D2 RNAylated with NAD-capped RNAI were combined and incubated at 4 °C in a Rotary Mixer for 30 min. Entire samples were transferred to Mobicol mini spin columns. Beads were washed four times with 200 µl Ni-NTA wash buffer and subsequently eight times with 200 µl Strep wash buffer (50 mM Tris-HCl pH 7.5, 8 M urea). Beads were equilibrated in standard ligation buffer (10 mM MgCl2, 50 mM Tris-HCl pH 7.4) and blocked with BSA prior to 3'-RNA-adapter ligation at 4 °C overnight in the presence of standard ligation buffer, 50 mM β-mercaptoethanol, 0.05 µg/µl BSA, 15 % (v/v) DMSO, 5 µM adenylated RNA-3'-adapter, 0.5 U/µl T4 RNL1 (NEB) and 10 U/µl T4 RNL2, tr. K227Q (NEB). Protein was rebound by addition of NaCl to 1.5 M and incubation at 20 °C, 400 rpm for 20 min. Beads were subsequently washed six times with strep wash buffer and equilibrated in first strand buffer (50 mM Tris-HCl pH 8.3, 3 mM MgCl2, 75 mM KCl) and blocked with BSA. Reverse transcription of protein-bound RNA was performed in a 30 µl scale for 1 h at 40 °C using 10 U/µl Superscript IV Reverse Transcriptase (Invitrogen) in the presence of 5 µM RT primer, first strand buffer, 25 mM β-mercaptoethanol, 0.05 µg/µl BSA and 0.5 mM dNTPs. After incubation, NaCl was added to 1.5 M and incubated at 20 °C, 400 rpm for 1 h in order to rebind RNA-cDNA hybrids. Beads were subsequently washed five times with 0.25 x strep wash buffer (2 M urea, 50 mM Tris-HCl pH 7.5) and equilibrated in aExoI buffer (10 mM Tris-HCl pH 7.9, 5 mM β-mercaptoethanol, 10 mM MgCl2, 50 mM NaCl) and blocked with BSA. Residual RT primer was removed by ExoI digest with 1 U/µl E. coli ExoI (NEB) in ExoI buffer at 37 °C for at least 30 min. Beads were finally washed with 200 µl 0.25 x strep wash buffer five times and 200 µl immobilization buffer (10 mM Na-HEPES pH 7.2, 1 M NaCl) three times. cDNA was eluted by incubation of beads in 100 µl 150 mM NaOH at 55 °C for 25 min and by washing with 100 µl MQ water. Eluate pH was neutralized by addition of 0.05 volumes 3 M NaOAc pH 5.5. cDNA was removed from residual protein by phenol-chloroform extraction and precipitated with 2.5 volumes ethanol in the presence of 0.3 M NaOAc pH 5.5 overnight. Precipitated cDNA was C-tailed using 1 U/µl TdT (Thermo Fisher) in the presence of 1.25 mM CTP and 1 x TdT buffer at 37 °C for 30 min and subsequently inactivated at 70 °C for 10 min. 5 µM cDNA anchor (hybridization of fwd and rev anchor) were ligated to C-tailed cDNA in standard ligation buffer in the presence of 10 µM ATP and 1.5 U/µl T4 DNA Ligase (Thermo Fisher Scienttific) at 4 °C overnight. Ligation reactions were inactivated at 70 °C for 10 min and cDNA was ethanol-precipitated. For the preparation of the Illumina RNAylome Seq library, cDNA was amplified by PCR using 2 U Phusion Polymerase (Thermo Fisher Scientific) in the presence of 5 % (v/v) DMSO, 200 µM dNTPs and 2500 nM NEBNext Universal and Index Primer each (Primer Set 1, NEB). PCR products were purified by native PAGE purification and ethanol-precipitated. dsDNA concentration was determined via Quantus fluorometer (Promega) and library size was determined with the Bioanalyzer (Agilent). Equimolar amounts of each library were sequenced on a MiniSeq system (Illumina) using the MiniSeq High-Output Kit (150 cycles, Illumina) generating 20 million 151 bp single-end reads.