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Charles University in Prague, Faculty of Science, Department of Cell Biology

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Namespace:Charles University in Prague, Faculty of Science, Department of Cell Biology

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Protocols: Five hundred millilitre cultures of an untagged strain and strains expressing TAP-tagged CSL proteins were grown in the rich YES medium (0.5% yeast extract, 3% glucose, SP supplements (Formedium)) at 32 degrees C to OD 1.0, fixed with 1% formaldehyde for 30 min, quenched with 125 mM glycine, washed with PBS and broken with glass beads in Lysis Buffer (50 mM Hepes pH 7.6, 1 mM EDTA pH 8.0, 150 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate, FY protease inhibitors (Serva)) using the FastPrep24 machine (MP Biomedicals). A. Chromatin Immunoprecipitation protocol A.2 Preparing extract and setting up immunoprecipitations A.2.1 Thaw cross-linked cell pellet on ice for 5 min. Resuspend pellets (you have 2 eppendorf tubes per sample) in total of 800 microlitres ice-cold Lysis buffer (add 750 microlitres of LB) containing 2x protease inhibitors (from Roche EDTA-free), 1 mM PMSF. A.2.3 Prepare a 4L bucket filled with ice and ice-cold dH2O. You will need it for sonication. A.2.3 Pour 500-600 microlitres of acid-washed glass beads and break in FastPrep 9-12x20sec at 5.5 with 5min incubations on ice in between (DO the breaking in 2 ml screw-cap tubes). Visualize a small aliquot in the microscope of cells to estimate the breaking efficiency (should be >90%). A.2.4 Poke a hole in the bottom of the tube with broken cells with a sterile needle after flaming it and place in a clean tube. Put the 2 tubes in a 50ml falcon and spin for 1min at 1000 rpm at 4ºC to collect the lysate. Wash the glass beads again with 400 microlitres lysis buffer (with inhibitors), spin and pool with the first flow-through (the additional washing of beads increases yield). A.2.5 Spin the lysate for 10min at 20000rcf, pour off supernatant and wash once more with 800 microlitres lysis buffer (with inhibitors) (the additional wash increases yield). A.2.6 After the wash, resuspend the pellet in total 750 microlitres of cold Lysis buffer and pool both eppendorf tubes into one 2 ml tube (you’re back to 1 Eppendorf tube / sample). Re-add PMSF to 1 mM. Split into 5x 300 microlitre aliquots in Axygen 1.5 ml tubes (this is the volume and tubes for which sonication was calibrated). A.2.7 Sonicate 6 x 5min with 30sec ON/ 30sec OFF at HIGH setting, refill the water bath with cold water (no crushed ice chips!!) after each 5min interval (Bioruptor settings). This procedure will produce fragments chromatin fragments of ~200bps. The conditions of sonication have typically to be set-up in each lab. The volume and concentration of the extract as well as the duration and strength of the sonication will affect the size of the sheared chromatin. A.2.8 Spin the sonicated material at 20000rcf for 10min at 4 degrees C and collect the supernatant (chromatin extract, CE). Store the CE at -70 degrees C. Use 5 microlitres CE to check protein concentration / Bradford (should get > 10 mg/ml) Use 50 microlitres CE to check sonication efficiency (in screw-cap tubes): Add 150 microlitres TES, incubate 65 degrees C, O/N. Add 200 microlitres TE, 5 microlitres of DNase-free RNase (0.5 mg/ml), incubate 37 degrees C, 60min. Add 7 microlitres proteinase K (20 mg/ml), incubate 55 degrees C, 2 hours. Add 400 microlitres phenol/chloroform/IAA (25:24:1), vortex 20 sec, spin full speed, 10 min, 4 degrees C. Collect the upper water phase (~400 microlitres) in a 1.5 ml screwcap tube. Re-add 100 microlitres TE to the lower organic phase, vortex 20 sec, spin full speed, 10 min, 4 degrees C (this increases yield). Collect the upper water phase and pool with the first one (~500 microlitres in total). Add 500 microlitres chloroform/IAA, vortex 5 sec, spin full speed, 5 min, 4 degrees C. Collect the upper water phase. Add 1 microlitre glycogen 20 mg/ml, 50 microlitres NaAc 3M, 1.25 ml ice-cold EtOH 100%. Incubate -70 degrees C, 30 min. Spin full speed, 15 min, 4 degrees C. Wash with 1ml ice-cold EtOH 70% (caution: loose pellet), spin 5 min 4 degrees C. Dry pellet in speedvac and resuspend in 10 microlitres water. Analyze 1 microlitre on Bioanalyzer or load everything on agarose 1%, 80V, EtBr stain. These samples can be used as 'input' for library construction. A.3 Immunopreciptitations, crosslink reversal and DNA purification. A.3.1 Keep 50 microlitres of CE for input (it can be stored at -20C). A.3.2 Prepare magnetic beads (all incubations and washes at 4 degrees C): Add 100 microlitres magnetic beads (Protein G-coated for rabbit/goat polyclonal Ab or mouse/sheep IgG-coated for TAP-tagged proteins, Invitrogen, Dynabeads) to a 1.5 ml microfuge tube. Add 1 ml block solution (0.5% BSA (w/v) in LB). Set up 1 tube per IP. Collect the beads using magnetic stand. Remove supernatant by aspiration. Wash beads in 1.0 ml block solution two more times. Resuspend beads in block solution and add 5 micrograms of antibody in a final volume of 250 microlitres. Incubate overnight or a minimum of 4 h on a rotating platform at 4 degrees C. Wash magnetic beads as described above (3 times in 1 ml block solution). Resuspend in 50 microlitres block solution. A.3.3 Use 5 mg CE (from Bradford) per sample, per IP. Bring up to 500 microlitres with LB (without inhibitors). A.3.4 Add the 50 microlitres antibody/magnetic bead mix to cell lysates. Gently mix at 4 degrees C in rotating wheel overnight. A.3.5 Collect the beads using magnetic stand. Remove supernatant by aspiration. Wash beads in 0.8ml of each buffer in semi-cold conditions: 2x in Lysis buffer 2x in Lysis 500 2x in LiCl/NP-40 1x in TE. A.3.6 Resuspend the beads in 200 microlitres elution buffer. Elute and perform reverse crosslinking at 65 degrees C for 6-18 h. Resuspend beads in the first 15 min with brief vortexing every 5 min. A.3.7 For the input, add 150 microlitres (3x the volume) of TES, also perform reverse crosslinking at 65 degrees C for 6-18 h and treat subsequently as the samples. The volume is increased to dilute out the SDS. A.3.8 Digest proteins and RNA: Add 200 microlitres TE, 5 microlitres of DNase-free RNase (0.5 mg/ml), incubate 37 degrees C, 60min Add 7 microlitre proteinase K (20 mg/ml), incubate 55 degrees C, 2 hours. The RNA is removed because it may interfere with the labelling reaction. A.3.9 Purify DNA (in screw-cap tubes): Add 400 microlitres phenol/chloroform/IAA (25:24:1), vortex 20 sec, spin full speed, 10 min, 4 degrees C. Collect the upper water phase (~400 microlitres) in a 1.5 ml screwcap tube. Re-add 100 microlitres TE to the lower organic phase, vortex 20 sec, spin full speed, 10 min, 4 degrees C (this increases yield). Collect the upper water phase and pool with the first one (~500 microlitres in total) Add 500 microlitres chloroform/IAA, vortex 5 sec, spin full speed, 5 min, 4 degrees C. Collect the upper water phase to a 2 ml tube. Add 1 microlitre glycogen 20 mg/ml, 50 microlitres NaAc 3M, 1.25 ml ice-cold EtOH 100% Incubate -70 degrees C, 1-2 h. Spin full speed, 15 min, 4 degrees C, process only 4 samples at a time for the next steps. Remove EtOH (caution: loose and tiny tiny pellet) and wash with 1ml ice-cold EtOH 70% (caution: loose and tiny tiny pellet), spin 5 min 4 degrees C. Dry pellet in speedvac. Resuspend in 25 microlitres TE, incubate at RT for 10 min. Purify on a Qiagen column to get rid of low-molecular weight debris and run 1 microlitre on a Bioanalyzer. Store -20 degrees C. These DNA samples can be now l used as starting material for ChIP-seq library preparation! Buffers: Lysis Buffer: 50mM Hepes pH 7.6 1mM EDTA pH8 150mM NaCl 1% Triton X-100 0.1% Na-Doc Protease Inhibitors; Lysis 500 Buffer: 50mM Hepes pH 7.6 1mM EDTA pH8 500mM NaCl 1% Triton X-100 0.1% Na-Doc; LiCl/NP-40 Buffer: 10mM Tris-HCl pH8 1mM EDTA pH8 250mM LiCl 1% NP-40 1% Na-Doc; Elution Buffer: 50mM Tris-HCL pH8 10mM EDTA 1% SDS; TE Buffer: 10mM Tris-HCL pH8 1mM EDTA; TES Buffer: 10mM Tris-HCL pH8 1mM EDTA 1% SDS Perform end-repair of the DNA use the End-it kit (Epicentre Biotechnologies, ER0720) 1. Mix together (total reaction volume 50 microlitres): 150 ng DNA in 34 microlitres total (see Nanodrop), 5 microlitres End-It 10X buffer, 5 microlitres ATP (10 mM), 5 microlitres dNTPs mix (2.5 mM), 1 microlitre End-It enzyme mix. 2. Incubate for 1 hour at RT. 3. Clean up samples using the DNA Clean&Concentrator-5 kit (Zymo Research), elute with 3x 11 microlitres water preheated to 50 degrees C. 4. Chill on ice. Add “A” bases to the DNA 1. Mix together (total reaction volume 50 microlitres): 32 microlitres DNA eluate, 5 microlitres Klenow 10X buffer (NEB, B7002S), 10 microlitres dATP (1 mM), 3 microlitres Klenow 3'-5' exo- (NEB, M0212L). 2. Incubate for 30 min at 37 degrees C. 3. Clean up samples using the DNA Clean&Concentrator-5 kit (Zymo Research), elute with 9 microlitres water preheated to 50 degrees C (incubate columns 1 min at RT before spinning). 4. Chill on ice. Ligate sequencing adapters to DNA fragments. Use adapters from the Illumina Multiplexed PE Sample Preparation Kit (1005361). 1. Mix together (total reaction volume 25 microlitres): 8 microlitres DNA eluate, 12.5 microlitres Quick Ligation Reaction Buffer (NEB, M2200L), 2 microlitres of 40x diluted Index PE Adapter Oligo Mix (Illumina), 2.5 microlitres Quick T4 DNA Ligase (NEB, M2200L). 2. Incubate for 15 min at RT. 3. Clean up samples using the DNA Clean&Concentrator-5 kit (Zymo Research), elute with 3x 15 microlitres water preheated to 50 degrees C. 4. Chill on ice. 5. Use 22 microlitres of the eluate for PCR and keep the other 22 microlitres as backup. Amplify adapter-modified DNA by PCR. Use primers from the Illumina Multiplexed PE Sample Preparation Kit (1005361). 1. Mix together in PCR tubes (total reaction volume 50 microlitres): 22 microlitres DNA eluate, 25 microlitres Phusion Master Mix with HF Buffer (NEB, F-531L), 1 microlitre PCR Primer InPE 1.0 (Illumina), 1 microlitre PCR Primer InPE 2.0 (Illumina), 1 microlitre PCR Primer Index<2/4/5/6/7/12> (Illumina). 2. Run PCR program: 98 degrees C 30 sec, 98 degrees C 10 sec, 65 degrees C 30 sec, 72 degrees C 30 sec, goto step 2, 17 times, 72 degrees C 5 min, 4 degrees C hold. 3. Clean up samples using the AMPure XP beads (Agencourt) - two successive rounds of purification are employed to get rid of DNA <100 bp and >300 bp. Place the beads at RT for 30 min before using them (do not use QiaQuick columns as they do not remove primers and adaptor dimers efficiently): “negative beads” 1. Mix AMPure XP beads with the PCR reaction in a 0.8 (beads) to 1.0 ratio (DNA) in 1.5 ml tubes. Under such conditions the beads selectively bind only higher molecular weight (>300 bp) fragments. 2. Incubate 5 min at RT. 3. Place tubes on a magnetic stand for 2 min. 4. Collect flow-through (this contains your DNA!!!). “positive beads” 1. Mix AMPure XP beads with the flow-through from the previous step in a 1.8 (beads) to 1.0 ratio (DNA) in 1.5 ml tubes. Only fragments >100 bp are bound (removes primers and also minimizes the adapter dimer content!). 2. Incubate 5 min at RT. 3. Place tubes on a magnetic stand for 2 min. 4. Keep the tubes on the stand. Remove flow-through. 5. While still on the stand, wash the beads with 70% EtOH (gently tilt the stand to allow the EtOH to reach the beads) for 30 sec. 6. Remove the EtOH and repeat the wash once again. 7. Open the tubes and let the beads air-dry for <5 min at RT (do not overdry - manifests as cracks in the bead mass). 8. Remove the tubes from the stand and elute with 40 microlitres water (mix by pipetting) for 1 min (do not use pre-heated water for elution as this might denature the library, which needs to remain double-stranded for sequencing). 9. Place tubes on a magnetic stand for 2 min. 10. Collect the eluate in a new tube and keep on ice. Check library quality on Bioanalyzer. Use DNA 1000 chips for high-concentration samples (inputs) and DNA High sensitivity chips for low-concentration samples (IPs).