GSM5412601: Wild-type nascent RNA-Seq biological replicate 3; Saccharomyces cerevisiae; RNA-Seq
For nascent RNA sequencing (nascent RNA-seq), RNA fragments associated with RNA polymerase II subunit Rpb3 endogenously tagged with 3xFLAG epitope at the C-terminus were isolated by affinity purification. Small batch cultures of yeast cells grown in YPD media were collected by centrifugation, the supernatant was aspirated, and cell pellets were immediately snap-frozen by submerging in liquid nitrogen to minimise changes in nascent transcription activity (e.g. in response to cell resuspension in cold lysis buffer with high concentrations of salts and detergents). Frozen cell pellets were dislodged from centrifuge tubes and stored at -80 °C. Cells were subjected to cryogenic lysis by freezer mill grinding under liquid nitrogen (SPEX 6875D Freezer/Mill, standard program: 15 cps for 6 cycles of 2 minutes grinding and 2 minutes cooling each). Yeast “grindate” powder was stored at -80 °C. 2 g of yeast grindate was resuspended in 10 mL of 1X cold lysis buffer (20 mM HEPES pH 7.4, 110 mM potassium acetate, 0.5% v/v Triton-X-100, 1% v/v Tween-20) supplemented with 10 mM MnCl2, 1X Roche cOmplete EDTA-free protease inhibitor, and 50 U/mL SUPERase.In RNase Inhibitor. Chromatin-bound proteins were solubilised by incubation with 1320 U of DNase I (RQ1 RNase-free DNase I, Promega) on ice for 20 minutes. Lysates containing solubilised chromatin proteins were clarified by centrifugation at 20,000 x g for 10 minutes (at 4 °C), and the supernatant was taken as input for immunoprecipitation using 500 μL of anti-FLAG M2 affinity gel suspension (A2220, Sigma-Aldrich) per sample (2.5 hours at 4 °C). After immunoprecipitation, the supernatant was removed and beads were washed 4 times with 10 mL of cold wash buffer each time (1X lysis buffer with 50 U/mL Superase.In RNase inhibitor and 1 mM EDTA). After the last wash, agarose beads were transferred to small chromatography spin columns (Pierce Spin Columns, Thermo Fisher), and competitive elution of protein complexes containing Rpb3-3xFLAG protein from the resin was performed by incubating beads with 300 μL of elution buffer (1X cold lysis buffer with 2 mg/mL 3xFLAG peptide) for 30 minutes at 4 °C (3xFLAG peptide provided by the Peptide Chemistry Science Technology Platform, The Francis Crick Institute). Elution was performed twice and 600 μL of eluate was subjected to acid phenol:chloform RNA extraction and ethanol precipitation. A significant amount of 3xFLAG peptide co-precipitates with the RNA as a contaminant, and is later removed by spin column purification. Purified RNA was fragmented to a mode length of ~200 nucleotides using zinc ion-mediated fragmentation (Ambion AM870, 70 °C for 4 minutes). Fragmented RNA was purified using miRNeasy spin columns (miRNeasy mini kit, QIAGEN), which retain RNAs approximately 18 nucleotides or more in length. Purified RNA was quantified by Qubit (Thermo Fisher) and approximately 150 ng of RNA was subjected to rRNA depletion using the Ribo-Zero Gold rRNA Removal Kit (Yeast) (Illumina MRZY1324, now discontinued). Libraries were prepared using the TruSeq Stranded Total RNA kit (Illumina) according to the manufacturer's instructions (14 PCR cycles). The libraries were multiplexed and sequenced on the HiSeq 4000 platform (Illumina), and generated ~28 million 101 bp strand-specific paired-end reads per sample on average.