Illumina HiSeq 2000 sequencing; TEF-seq/NET-seq applied to factors Rpb3, Spt6, Spt16, Cet1, Paf1, Pcf11, Ssu72, Set2.
TEF-seq/NET-seq applied to factors Rpb3, Spt6, Spt16, Cet1, Paf1, Pcf11, Ssu72, Set2.
Cells were grown in 25 ml YPD (1 % yeast extract (Difco), 1 % bactopeptone, 2 % glucose) overnight. This was used to inoculate 1 L of YPD in a 2 L flask to give a starting OD600 of 0.2. 2 L of cells were grown (30oC, 170 rpm) to OD600 of 0.65 (mid-log) and harvested by filtration onto a 0.45 µm pore size, 90 mm diameter nitrocellulose filter paper (Whatman). Cells were scraped off the filter paper with a spatula pre-cooled in liquid nitrogen and flash frozen in liquid nitrogen. Cells were ground in 6 cycles of 3 min at a 15 Hz shaking frequency in a 50 ml grinding jar (Retch) with a 25 mm stainless steel ball using a Retsch MM400 mixer mill. The grinding chamber was cooled in liquid nitrogen in between cycles. 1 g of yeast grindate was stored at -80 oC.  For carbon source shift experiments 2 L of cells were grown to mid-log in 2 x 2 L flasks, then centrifuged (3000 rpm, 3 min) and then resuspended in 2 L YPG (1 % yeast extract (Difco), 1 % bactopeptone, 2 % galactose) prewarmed to 30oC. Resuspended cells were returned to 2 L flasks, incubated (30oC, 170 rpm) and harvested by filtration as above after the requisite incubation time. 1 g of yeast grindate was resuspended in Lysis Buffer A (20 mM HEPES (pH 7.4), 110 mM KOAc, 0.5 % Triton-X-100, 0.1 % Tween 20, 10 mM MnCl2, 1x proteinase inhibitors (complete, EDTA-free (Roche)), 50 U/ml SUPERase.In (Invitrogen)). Resuspended grindate was incubated (4 oC, 20 min) with 660 U of DNase I (Promega). Insoluble cell debris was pelleted by spinning (16,000 g, 4 oC, 10 min). Supernatants were combined and added to 0.5 ml of anti-FLAG M2 affinity agarose beads (Sigma) pre-washed with 2 x 10 ml Lysis buffer A (without SUPERase.In). Beads and supernatant were incubated (4 oC, 2.5 h) on a nutator, before spinning (1,000 g, 4 oC, 2 min). Beads were washed four times with 10 ml Wash Buffer A (20 mM HEPES (pH 7.4), 110 mM KOAc, 0.5 % Triton-X-100, 0.1 % Tween 20, 1 mM EDTA). Beads were incubated (30 min, 4 oC) twice with 300 µl of Elution Buffer (Lysis Buffer A with 1 mg/ml 3 x FLAG peptide (Sigma)). The elution supernatants were combined and RNAs were extracted using a miRNeasy kit (Qiagen) according to the manufacturer’s instructions. For the sequential IP procedure, the elution supernatants from two IPs were combined giving a total of 1200 ul and added to 15 ul anti-HA dynabeads (Pierce) pre-washed with 2 x 1 ml Lysis buffer A. Beads and supernatant were incubated (4 oC, 2.5 h) on a nutator. Beads were washed five times with 1 ml Wash Buffer A. Beads were resuspended in 100 ul Lysis buffer A and RNAs were extracted using a miRNeasy kit (Qiagen) according to the manufacturer’s instructions. Library generation steps: Linker ligation and RNA fragmentation. Immunoprecipitated RNA (3 μg in 30 µl 10 mM Tris-HCl, pH 7.0) was denatured (2 min, 80 oC) and placed on ice. A 5’ adenylated, 3’ blocked with a dideoxy C base cloning linker 5rApp/CTGTAGGCACCATCAAT/3ddC (Integrated DNA Technologies) was ligated to the 3’ ends of RNAs by first dividing the RNA into three microfuge tubes and then adding 10 µl ligation reaction mix to give final concentrations of 50 ng µl-1 cloning linker 1, 12 % PEG 8000, 1 x T4 RNA ligase 2 (Rnl2) (truncated) ligation buffer, 10 U µl-1 T4 Rnl2 (truncated) (NEB) and incubating for 3 h at 37 oC. Ligated RNA was incubated (95 oC, 35 min) with 20 µl Alkaline Fragmentation Buffer (100 mM NaCO3 (pH 9.2), 2 mM EDTA) to fragment linker ligated RNA to a narrow size range to reduce size bias of future steps. RNA was precipitated by incubating (30 min, -20 oC) with ice cold 500 µl H20, 60 µl 3 M NaOAc (pH 5.5), 2 µl 15 mg ml-1 GlycoBlue (Ambion) and 0.75 ml isopropanol and then spinning (16,000 g, 4 oC, 30 min). Pellets were washed with 0.75 ml 80 % ethanol, dried (10 min, room temperature) and resuspended sequentially in the same 10 µl 10 mM Tris-HCl (pH 7.0). Size selection. Ligated and fragmented RNA was mixed with 10 µl 2 x TBE-urea loading dye (89 mM Tris-HCl, 89 mM Boric acid, 2 mM EDTA, 12 % Ficoll, 7 M Urea, 2.5 mg/ml Orange G), denatured (2 min, 80 oC) and run on a 10 well 10 % TBE-urea gel (Biorad) (200 V, 32 min). The gel was stained with SYBRGold (Invitrogen) and the region containing 38-95nt fragments excised. This gel piece was divided between three microfuge tubes and physically disrupted. Each was incubated (70oC, 10 min) in 200 µl H20. The tubes were pooled and gel debris was removed using a Costar-Spin-X column (Corning). RNA was precipitated by adding 60 µl 3 M NaOAc (pH 5.5), 2 µl 15 mg ml-1 GlycoBlue and 0.75 ml isopropanol, incubating (30 min, -20oC) and then spinning (16,000 g, 4oC, 30 min). Pellets were washed with 0.75 ml 80 % ethanol, dried (10 min, room temperature) and resuspended in 10 µl 10 mM Tris-HCl (pH 7.0). Reverse transcription and circularisation. Reverse transcription (RT) was carried out by adding 3.28 µl 5 x FS buffer, 0.82 µl dNTPs (10 mM each), 0.5 µl 100 µM RT primer (5’/5Phos/ATCTCGTATGCCGTCTTCTGCTTG/iSp18/CACTCA/iSp18/TCCGACGATCATTGATGGTGCCTACAG 3’; This is phosphorylated at the 5’ end (5 Phos) for later circularization and contains two 18 carbon spacer sequences (iSp18)), denaturing (80 oC, 2 min) and incubating (48oC, 30 min) with 0.5 µl Superase.In, 0.82 µl 0.1 M DTT, 0.82 µl Superscript III (Invitrogen). 1.8 µl 1 M NaOH was added and RNA was degraded (98 oC, 20 min). To neutralize, 1.8 µl 1 M HCl was added. cDNA was mixed with 20 µl 2 x TBE-urea loading dye, denatured (3 min, 95 oC), and run (loaded in 2 wells, 20 µl per well) on a 10 well 10 % TBE-urea gel (Biorad) (200 V, 50 min). The gel was stained with SYBRGold (Invitrogen) and the regions containing the RT product excised. Both gel pieces were physically disrupted and incubated (70 oC, 10 min) in 200 µl H20 in separate tubes. Gel debris was removed and tubes were pooled. cDNA was precipitated by adding 25 µl 3 M NaCl, 2 µl GlycoBlue and 0.75 ml isopropanol, incubating (-20 oC, 30 min) and then spinning (16,000 g, 4 oC, 30 min). The pellet was washed with 0.75 ml 80 % ethanol, dried (10 min, room temperature) and resuspended in 15 µl 10 mM Tris-HCl (pH 8.0). The RT product was circularized by incubating (60 oC, 60 min) with 2 µl 10 x CircLigase buffer, 1 µl 1 mM ATP, 1 µl 50 mM MnCl2, 1 µl CircLigase (Epicentre). The enzyme was heat-inactivated at 80 oC, 10 min. PCR amplification. 16.7 µl 5 x HF Phusion buffer, 1.7 µl  dNTPs (10 mM), 0.4 µl 100 µM Barcoded primer (A, AATGATACGGCGACCACCGAGATCTACACGATCGGAAGAGCACACGTCTGAACTCCAGTCACATGCCATCCGACGATCATTGATGG ;B, AATGATACGGCGACCACCGAGATCTACACGATCGGAAGAGCACACGTCTGAACTCCAGTCACTGCATCTCCGACGATCATTGATGG; C, AATGATACGGCGACCACCGAGATCTACACGATCGGAAGAGCACACGTCTGAACTCCAGTCACTTAGGCTCCGACGATCATTGATGG; or D, AATGATACGGCGACCACCGAGATCTACACGATCGGAAGAGCACACGTCTGAACTCCAGTCACTGACCATCCGACGATCATTGATGG;), 0.4  µl 100 µM Primer 1 (CAAGCAGAAGACGGCATACGA), 59.2 µl H20, 0.8 µl Phusion polymerase (NEB) was added to 5 µl circularized DNA. This was divided (16.7 µl per tube) between five 0.2 ml tubes. PCR reactions were heated (98oC, 30 s) and then submitted to 7 temperature cycles (98oC, 10 s; 60oC, 10 s; 72oC, 10 s). At the end of cycle 3 and each subsequent cycle one tube was removed and placed on ice. 3.4 µl DNA loading dye (1.5 g Ficoll 400, 25 mg Orange G in 10 ml H20) was added to each reaction and run on an 8 % TBE gel (Invitrogen) (180 V, 55 min). The gel was stained with SYBRGold and the PCR product excised from the PCR reaction with the highest unsaturated signal without higher molecular weight products. The gel piece was physically disrupted and incubated (room temperature, overnight, with agitation) in 0.67 ml DNA soaking buffer (0.3 M NaCl, 10 mM Tris-HCl pH 8.0, 1 mM EDTA). DNA was precipitated by adding 2 µl GlycoBlue and 0.68 ml isopropanol, incubating (-20oC, 30 min) and spinning (16,000 g, 4oC, 30 min). The pellet was washed with 0.75 ml 80 % ethanol, dried (10 min, room temperature) and resuspended in 10 µl 10 mM Tris-HCl (pH 8.0).
Experimental Factor: paf1, Non-FLAG-tagged
genotype
Experimental Factor: Glucose (YPD)
growth condition