GSM5694121: Wild-type, G1 phase (t=0 min), Input, Rep 1; Saccharomyces cerevisiae; OTHER
RNA was extracted using TRIzol (Thermo Fisher, #15596) following the manufacturer's instructions. Briefly, frozen cell pellets were resuspended in TRIzol and lysed using glass beads and a bead beater (Biospec products). RNA was extracted twice with chloroform, precipitated with isopropanol, washed twice with 75% ethanol and resuspended in H2O. RNA was stored at -80°C. Cell cycle progression was monitored by RT-PCR analysis, as described below, of HTA1 (histone H2A) and SWI5 genes whose expression peaks in S phase and G2/M respectively (data not shown). RNAs from t=0 min (G1 phase) and t=40 min (S phase) were selected for further polyadenylated tail enrichment and sequencing.  Polyadenylated (poly(A)) RNA was enriched using Oligo d(T)25 Magnetic Beads (NEB, #S1419S) following the manufacturer's instructions, using ~150 μg of purified total RNA.  Poly(A)-enriched RNA was treated with DNase I to remove any contaminating DNA using the RNA Clean & Concentrator kit (Zymo, #R1014) according to the manufacturer's instructions. RNA was ligated to the universal miRNA cloning linker (NEB, #S1315S) by combining 100 ng of poly(A)-enriched RNA with 1 μM universal miRNA cloning linker, 1X T4 RNA ligase buffer, 20% PEG 8000, 100 U T4 RNA ligase 2 (truncated KQ) (NEB, #M0373S), and 10 U SUPERase-In RNase inhibitor (ThermoFisher Scientific, #AM2694) in a 20 μL  volume and incubating at 25°C for 2 hr in a thermocycler. The linker-ligated RNA was purified using the RNA Clean & Concentrator kit (Zymo, #R1014) and eluted in 15 μL RNase/DNase-free water. The RNA was reverse-transcribed into cDNA using the SMARTer PCR cDNA Synthesis Kit (Clontech, #634925) with modifications to the manufacturer's protocol. Briefly, 10-50 ng of RNA was mixed with 1 mM dNTP mix and 1 μM of the following oligo: 5'- AAGCAGTGGTATCAACGCAGAGTNNNNNNATTGATGGTGCCTACAG-3', where N is either an A, C, G, or T, and the string of Ns creates a unique molecular identifier (UMI) that serves as a barcode for each cDNA molecule. This mixture was heated at 65°C for 2 min on a thermocycler and then combined with a pre-heated mixture of 1x First-Strand Buffer, 10 μM DTT, 1.2 μM Clontech SMARTer IIA template-switch oligo, 5 U SUPERase-In RNase inhibitor, and 100 U Maxima H Minus Reverse Transcriptase (ThermoFisher Scientific, #EP0751). This 10 μL mixture was incubated at 50°C for 1 hr, 45°C for 30 min, and 85°C for 5 min before being cooled on ice. The resulting cDNA was purified using the Monarch PCR & DNA Cleanup Kit (NEB, #T1030S) and amplified via PCR with 1x KAPA HiFi HotStart ReadyMix (Roche, #KR0370) and 1 μM of the following oligo: 5'-AAGCAGTGGTATCAACGCAGAGT-3' for 12 PCR cycles. The amplified DNA was purified with the QIAquick PCR Purification Kit (Qiagen, #28104) and eluted in 30 μL Elution Buffer.  To capture centromeric cDNA molecules, the xGen Hybridization and Wash Kit (IDT, # 1080577) was used with some modifications to the manufacturer's protocol. Briefly, 500 ng-1 μg of cDNA was lyophilized with 6 μM 5' blocking oligo (Clontech SMARTer IIA template-switch oligo), 6 μM 3' blocking oligo (Pacbio 3' blocker-16bpBC), and 1 μg salmon sperm (Invitrogen, #15-632-011) using a SpeedVac system. The lyophilized DNA was resuspended in 17 μL hybridization mastermix consisting of 8.5 μL xGen 2X Hybrization Buffer, 2.7 μL xGen Hybridization Buffer Enhancer, 1.8 μL water, and 4 μL of 0.75 μM biotinylated probes. The solution was heated at 80°C for 5 min, incubated at 95°C for 1 min, and then incubated at 65°C for 2-4 hours. Washed streptavidin beads (Invitrogen, #65306) were added to the mixture and incubated at 65°C for 45 min. The beads were washed with a series of heated and room temperature washes according to the manufacturer's protocol. The remaining DNA on the beads were amplified via PCR with 1x KAPA HiFi HotStart ReadyMix (Roche, #KR0370) and 1 μM of the following oligo: 5'-AAGCAGTGGTATCAACGCAGAGT-3' for 15 PCR cycles according to the manufacturer's instructions. The resulting DNA was purified and checked via Agilent's BioAnalyzer System to ensure successful amplification. Sequencing libraries were prepared using the SMRTbell Express Template Prep Kit 2.0 (Pacific Biosciences, #100-938-900) according to the manufacturer's protocol. Briefly, 250 ng of DNA was damage-repaired by mixing it with 7 μL DNA prep buffer, 0.6 μL NAD, and 2 μL DNA Damage Repair Mix v2. This reaction was incubated at 37°C for 30 min before proceeding to the end-repair step. For the end-repair step, 3 μL End Prep Mix was added to the reaction and incubated at 20°C for 30 min, followed by a second incubation at 65°C for 30 min. The reaction was then mixed with 3 μL barcoded overhang adapter, 30 μL Ligation Mix, 1 μL Ligation Enhancer, and 1 μL Ligation Additive to ligate the barcoded adapter. This reaction was incubated at 20°C for 1 hr. The SMRTbell libraries were cleaned up with ProNex beads (Promega, #NG2001), pooled, and sequenced on a SMRT Cell 8M on the Pacific Biosciences Sequel II in CCS mode with the following parameters: --noPolish –minLength 10 –minPasses=3).