Schizosaccharomyces pombe
Protocols: At OD 0.5 10 ml aliquots of yeast cultures were centrifuged for 2 minutes at 600g. Supernatant was discarded. Pellets were flash-frozen in liquid nitrogen and placed in storage at -75°C. Cultures were pre-grown in YES to the exponentiation phase at 32°C. Then two aliquots of each cultures were centrifuged for 3' at 1000g, washed with YES and YES+93.5mM ammonium chloride (in parallel), diluted to OD=0.005 (WT) or 0.03 (cbf11Δ) and grown in corresponding media at 32°C. Caution: always use gloves, separate solutions and special tubes/tips when working with RNA 1. Harvest cells (usually 10 ml of OD600 ~0.5, adjust volume according to OD). Centrifuge 2' at 600 g in a falcon tube and discard supernatant (SN). Snap freeze pellet (liquid nitrogen or dry ice/ethanol). Alternatively, filter cells and snap freeze filter disc. Store cells at -80°C. 2. Thaw cells on ice (~5 min). Add 1 ml of ice-cold nuclease-free (NF) water, resuspend cells, and transfer to 2ml Eppendorf tubes. Spin 10'' at 2300 g and remove SN. Return tubes on ice.3. To pellet add 750 µl of TES (adjust if total cells are >5 ODs), resuspend cells with pipette, immediately add 750 µl acidic phenol-chloroform (refrigerated, Sigma P-1944 or P4682+C0549 5:1 mix; under fume hood), vortex, and place in 65°C heat block (use fume hood!). Only then process the next sample in the same way. 4. Incubate all samples in 65°C heat block for 1 hr, vortex 10'' every 10'. 5. Place samples on ice for 1', vortex 20'', and centrifuge for 15' at 20,000 g at 4°C. 6. Pre-spin 2ml MaXtract™ High Density tubes (QIAGEN, #129056; 1 tube per sample) for 10''. Add 700 µl of acidic phenol-chloroform (under fume hood). 7. Take 700 µl of the water phase from step 5 and add to the MaXtract™ tubes from step 6, thoroughly mix by inverting (no vortexing), and centrifuge 5' at 20,000 g at 4°C. 8. Pre-spin new 2ml MaXtract™ tubes as in step 6. Add 700 µl of chloroform:isoamyl alcohol (24:1, under fume hood, Sigma C-0549). 9. Take 700 µl of the water phase from step 7 and add to the MaXtract™ tubes from step 8, mix thoroughly by inverting (no vortexing), and centrifuge 5' at 20,000 g at 4°C. 10. Prepare normal 2ml Eppendorf tubes (1 tube per sample) with 1.5 ml of 100% EtOH and 50 µl of 3M NaAc pH 5.2. 11. Transfer 500 µl of water phase from step 9 to the tubes from step 10, vortex 10''. Samples can be precipitated at -20°C overnight (or at -80°C for 30 min). 12. Centrifuge for 10' at 20,000 g at RT. Discard SN, add 500 µl 70% EtOH (4°C, made with NF water), don't vortex, just add, and spin for 1' (same tube orientation!). Aspirate most SN, spin 5', and remove the rest of liquid with pipette. Air dry 5'' at RT. 13. Add 100 µl of NF water, and incubate 1' at 65°C. Dissolve pellet first by pipetting up and down (~30x) until no particles are left, then gently vortex 10''.14. Measure concentrations with NanoDrop.15. Treat with TURBO DNase (ThermoFisher AM2238, see appendix 1).16. Clean with RNeasy columns (QIAgen74104). Elution with TE (see appendix 2, note the differences from the kit manual!).17. OPTIONAL: Run 10 µl of purified RNA on a 1% agarose gel (wipe gel apparatus/tray with RNAse-Zap, rinse with water and use new TBE buffer, use RNAse-free loading buffer). You should see the two ribosomal bands clean, distinct and without smears.18. Send an aliquot to Qubit and Bioanalyzer - 5ul in strips.TES: 10mM Tris pH 7.5; 10mM EDTA pH 8; 0.5% SDS (store at RT)Appendix 1. RNase treatment1. Add 10ug of RNA according to NanoDrop data.2. Add 5ul of 10x TURBO DNase Buffer.3. Add 1ul (2U) of TURBO DNase.4. Adjust volume to 50ul with RNase-free water. Mix by pipetting.5. Incubate 37°C 30'. Immediately proceed with column purification.Appendix 2. Column purification1. Adjust the sample to a volume of 100 μl with RNAse-free water. Add 350 μl Buffer RLT, and mix well.2. Add 250 μl ethanol (96–100%) to the diluted RNA, and mix well by pipetting. Do not centrifuge. Proceed immediately to step 3.3. Transfer the sample (700 μl) to an RNeasy Mini spin column placed in a 2 ml collection tube (supplied). Close the lid gently, and centrifuge for 15 s at ≥8000 x g (≥10,000 rpm). Discard the flow-through. Reuse the collection tube in step 4.Note: After centrifugation, carefully remove the RNeasy spin column from the collection tube so that the column does not contact the flow-through. Be sure to empty the collection tube completely.4. Add 500 μl Buffer RPE to the RNeasy spin column. Close the lid gently, and centrifuge for 15 s at ≥8000 x g (≥10,000 rpm) to wash the spin column membrane. Discard the flow-through.Reuse the collection tube in step 5. Note: Ensure that ethanol is added to Buffer RPE before use.5. Add 500 μl Buffer RPE to the RNeasy spin column. Close the lid gently, and centrifuge for 2 min at ≥8000 x g (≥10,000 rpm) to wash the spin column membrane.Note: After centrifugation, carefully remove the RNeasy spin column from the collection tube so that the column does not contact the flow-through. Otherwise, carryover of ethanol will occur.6. Optional: Place the RNeasy spin column in a new 2 ml collection tube (supplied), and discard the old collection tube with the flow-through. Close the lid gently, and centrifuge at full speed for 1 min.Perform this step to eliminate any possible carryover of Buffer RPE, or if residual flow-through remains on the outside of the RNeasy spin column after step 5.7. Place the RNeasy spin column in a new 1.5 ml collection tube (supplied). Add 50 μl of TE buffer directly to the spin column membrane. Close the lid gently, leave for 5' at RT. Centrifuge for 1 min at ≥8000 x g (≥10,000 rpm) to elute the RNA.8. Collect the flow-through and load it back on the membrane. Centrifuge for 1 min at ≥8000 x g (≥10,000 rpm) to elute the RNA. The Illumina TruSeq stranded mRNA Library (poly-A) kit was used for library construction using the manufacturer's instructions.
ENA first public
2024-04-05
External Id
SAMEA114307710
INSDC center name
Charles University, Faculty of Science Department of Cell Biology
INSDC last update
2023-08-29T10:19:19Z
INSDC status
public
Submitter Id
E-MTAB-13302:Dcbf11_3
broker name
ArrayExpress
collection date
not collected
common name
fission yeast
genotype
Δcbf11::kanR, h+
geographic location (country and/or sea)
not collected
growth condition
YES medium
isolate
not applicable
organism part
whole organism
replicate
3
sample name
E-MTAB-13302:Dcbf11_3
scientific_name
Schizosaccharomyces pombe
strain
MP44