TITLE: ChIP-SICAP
AUTHORS: Sina Rafiee, Jeroen Krijgsveld

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Chromatin immunoprecipitation combined with mass spectrometry (ChIP-MS) suffers from unexpected proteins that bind to chromatin during the cell lysis and immunoprecipitation (IP). The so-called hitchhikers of chromatin are hardly washed away by gentle washing steps applied to a normal IP protocol. We employed DNA end-labeling by Terminal Transferase (TdT) to purify proteins specifically bound to chromatin. Using this double purification strategy, chromatin fragments are released from IP beads, and re-captured by streptavidin beads. Thus, we can apply stringent conditions to remove the hitchhikers of chromatin, and to identify authentic proteins co-localized with the protein of interest. We call this protocol Selective Isolation of Chromatin-Associated Proteins (SICAP). So far, ChIP-SICAP was successfully applied to different cell lines such mouse ES cells, mouse fibroblast cells, HEK 293T, HeLa, U2OS as well as mouse and human tissues. Here, we share the most updated version of our protocol. If you have any questions please feel free to email us.</div></div>

[STEPS]
?. [Part #1-A: detach and crosslink the cells ]
Detach the cells by Accutase. Accutase doesn’t lyse cells, so it is more suitable for single cell preparation. After detaching the cells, make sure that they are single cell by pipetting in the medium.
?. [Part #1-A: detach and crosslink the cells ]
Count the cells.
?. [Part #1-A: detach and crosslink the cells ]
Resuspend the cells in formaldehyde 1.5% in PBS. Roughly for every 10 million mammalian cells, add 10ml formaldehyde 1.5%. Pipette up and down to disrupt the cell pellet completely.
?. [Part #1-A: detach and crosslink the cells ]
Rotate occasionally, and keep it 15min at room temperature.
?. [Part #1-A: detach and crosslink the cells ]
Add Glycine 1M to stop the crosslinking, so the final concentration will be 130mM (1.5ml Glycine for every 10ml of formaldehyde).
?. [Part #1-A: detach and crosslink the cells ]
Rotate occasionally 5min at room temperature.
?. [Part #1-A: detach and crosslink the cells ]
Spin the cells at 2000RPM, 2min. Discard the supernatant (SN).
?. [Part #1-A: detach and crosslink the cells ]
Resuspend the cells with PBS-T, equal volume to the formaldehyde volume that you'd used in step 4
?. [Part #1-A: detach and crosslink the cells ]
Spin the cells at 2000RPM, 2min, and discard the SN.
?. [Part #1-A: detach and crosslink the cells ]
Resuspend the cells in some PBS-T
?. Inside a fume hood, remove the medium, and pour 10ml of formaldehyde 1.5% in a 10cm dish.
?. ·If you estimate there are more than 10 million cells, you may pour 20ml of formaldehyde
?. Wait 15min, and rotate the plate gently.
?. Add Glycine to the final concentration of 130mM
?. Wait 5min, and rotate the plate gently.
?. In the meantime, mix LB3 with 1x Complete protease inhibitor. For each 10cm plate you may need 10ml of LB3
?. Discard the liquid, and pour 10ml of PBS
?. Rotate the plate gently, and gently discard the liquid
?. Repeat the last two steps once again.
?. Pour 3ml of LB3+Complete on the plate.
?. ·LB3 allows to collect the cells more efficiently. While, using PBS you will lose quite some cells.
?. 10.Scrap the cells with LB3, and collect them in one side of the plate. Then collect them and pour them in a Falcone tube (15 or 50ml depending on the volume)
?. 11.Repeat the last two steps, twice.
?. 12.Spin the cells at 2000RPM for 2 min, and discard the SN
?. 13.Resuspend the cells in LB3+Complete
?. 14.Repeat the last two steps once again.
?. 15.To measure the protein concentration, take 5ul of the cells, pour it in a PCR tube and add 1ul of Benzonase
?. 16.Incubate at 37°C for 30min
?. 17.Add 30ul of SDS wash buffer, and incubate at 95°C for 5 min
?. 18.Measure the protein concentration by a BCA kit
?. ·The standards of the BCA should be treated the same. Namely, mix 5ul of the standards with 30ul of the SDS wash buffer, and incubate them at 95°C for 5min. Then 25ul of each sample/standard should be loaded on a 96w plate to measure the protein concentrations by the BCA reagents. A 2mg/ml standard and blank standard would be enough to compare the protein concentrations of the samples.
?. 19.Calculate the protein concentrationsAdjust the protein concentration with LB3+compele, aliquot the samples
?. [Part #1-A: detach and crosslink the cells ]
Count the cells, and Aliqoute them. e.g. every 24 millions in a 15-ml tube
?. [Part #1-A: detach and crosslink the cells ]
Spin the cells at 2000 RPM 2min, and discard the SN. Freeze the cells in liquid nitrogenPause point: You may freeze the cells in -80°C for months.
?. [Part #1-B: crosslinking the cells in a plate, and then harvesting them]
Wait 15min, and rotate the plate gently.
?. [Part #1-B: crosslinking the cells in a plate, and then harvesting them]
Add Glycine to the final concentration of 130mM
?. [Part #1-B: crosslinking the cells in a plate, and then harvesting them]
Rotate the plate gently, and wait 5min.
?. [Part #1-B: crosslinking the cells in a plate, and then harvesting them]
Discard the liquid,  gently pour 10ml of PBS on the cells, rotate, and discard it
?. [Part #1-B: crosslinking the cells in a plate, and then harvesting them]
Repeat the last  step once again.
?. [Part #1-B: crosslinking the cells in a plate, and then harvesting them]
Pour 3ml of PBS-T (Tween 0.1%) on the plate.Triton allows to collect the cells more efficiently. While, using PBS you will lose quite some cells.Adding Complete protease inhibitor is optional. Apparantely, endogenous proteases are inactivated by formaldehyde.
?. [Part #1-B: crosslinking the cells in a plate, and then harvesting them]
Scrap the cells with a cell lifter, and collect them in one side of the plate. Then transfer them by a pipet to a tube (e.g. 15ml or 50ml depending on the number of the plates)
?. [Part #1-B: crosslinking the cells in a plate, and then harvesting them]
Repeat the last two steps, once again for each plate.
?. [Part #1-B: crosslinking the cells in a plate, and then harvesting them]
Spin the cells at 2000RPM for 2 min, and discard the supernatant
?. [Part #1-B: crosslinking the cells in a plate, and then harvesting them]
Resuspend the cells in PBS-T, and aliqoute the them if you wish
?. [Part #1-B: crosslinking the cells in a plate, and then harvesting them]
Spin the cells at 2000RPM 2 min, and discard the supernatant
?. [Part #1-B: crosslinking the cells in a plate, and then harvesting them]
Freeze the cells in liquid nitrogen, and keep them in -80 ºCPause point: You may freeze the cells in -80°C for months.
?. [Part #1-B: crosslinking the cells in a plate, and then harvesting them]
Inside a fume hood, remove the medium, and pour 10ml of formaldehyde 1.5% in a 10cm dish.
?. [Part #1-A: detach and crosslink the cells ]
Spin the cells at 200g for 5min, and remove the medium.
?. [Part #2: Sonicating the cells and shearing chromatin]
1-Resuspend
?. [Part #2: Sonicating the cells and shearing chromatin]
million cells in 1ml of LB3+Complete and vortex, and divide it into 2x 1.5ml tubes specifically designed for Bioruptor pico
?. [Part #2: Sonicating the cells and shearing chromatin]
·
?. [Part #2: Sonicating the cells and shearing chromatin]
0pt;line-height:150%;font-family:"Arial",sans-serif'>Do not pour more than 12million cells in one tube for sonication.
?. [Part #2: Sonicating the cells and shearing chromatin]
2-Keep on ice for 5min
?. [Part #2: Sonicating the cells and shearing chromatin]
3-Spin 2000g, 2min
?. [Part #2: Sonicating the cells and shearing chromatin]
4-Discard the supernatant.
?. [Part #2: Sonicating the cells and shearing chromatin]
5-Resuspend the pellet in 500ul of LB3+ Complete, and repeat the steps 2 to 4, once again.
?. [Part #2: Sonicating the cells and shearing chromatin]
6-Resuspend each tube in 300ul of LB3 buffer.
?. [Part #2: Sonicating the cells and shearing chromatin]
7-Sonicating conditions depends on the cell lines. Bioruptor Pico is faster, however, Covaris is more precise and reproducible. You may start with Bioruptor Pico, and apply 7 cycles ON and OFF (30’’-30’’).
?. [Part #2: Sonicating the cells and shearing chromatin]
·If you used approach #2 for crosslinking, please thaw the cell lysates, and transfer 300ul to the sonication tubes. This should be about 1mg proteins as one replicate.
?. [Part #2: Sonicating the cells and shearing chromatin]
·Adding SDS to the final concentration of 0.1% improves the efficiency of sonication. However, some antibodies are sensitive to it.
?. [Part #2: Sonicating the cells and shearing chromatin]
?. [Part #2: Sonicating the cells and shearing chromatin]
8-After the sonication spin the sonication tubes 10min at 12000g
?. [Part #2: Sonicating the cells and shearing chromatin]
9-Collect the supernatant, and transfer it into a new 2ml epi-tube.
?. [Part #2: Sonicating the cells and shearing chromatin]
·
?. [Part #2: Sonicating the cells and shearing chromatin]
0pt;line-height:150%;font-family:"Arial",sans-serif'>You may take 5ul of the samples, to check the sheared chromatin. Boil and clean up DNA using QIAgen PCR purification kit. Run the DNA on an agarose gel. The smear should be
?. [Part #2: Chromatin immunoprecipitation (ChIP)]
Resuspend the cells in 0.9ml Tris-Cl 10mM. Then add 100ul Tritone X100 10%(v/v). Leave the cells on ice for 5 minOptional: You may also add Complete protease inhibitor.
?. [Part #2: Chromatin immunoprecipitation (ChIP)]
Spin the cells 2min at 2000RPM. Discard the supernatant.
?. [Part #2: Chromatin immunoprecipitation (ChIP)]
Resuspend the cells in 1ml LB3 buffer.  Spin the cells 2min at 2000RPM. Discard the supernatant.
?. [Part #2: Chromatin immunoprecipitation (ChIP)]
Repeat Step 31, twice again.
?. [Part #2: Chromatin immunoprecipitation (ChIP)]
Resuspend the cells in LB3 buffer.Resuspend  every 8 million cells in 250ul LB3 buffer, and-so-forth. One 10cm dish of confluent mES cells is about 20 million cells. Therefore, you may resuspend the cells in 750ul LB3 buffer.
?. [Part #2: Chromatin immunoprecipitation (ChIP)]
Sonicating conditions depends on the cell lines. For mES cells using Bioruptor Pico, you may apply 7 cycles ON and OFF (30’’-30’’).Bioruptor Pico is faster, however, Covaris is more precise and reproducible. Adding SDS to the final concentration of 0.1% improves the efficiency of sonication. However, some antibodies are sensitive to it. You may add 0.1% sodium deoxycholate or 0.1% Sarkosyl.Too many cycles of sonication blast the proteins from chromatin.
?. [Part #2: Chromatin immunoprecipitation (ChIP)]
After the sonication spin the sonication tubes 10min at 12000g
?. [Part #2: Chromatin immunoprecipitation (ChIP)]
Collect the supernatant, and transfer it into a 2ml epi-tube or a 15ml tube. You may pool the sheared chromatin obtained from one replicate.
?. [Part #2: Chromatin immunoprecipitation (ChIP)]
Measure the protein concentration using a BCA protein assay kit.
?. [Part #2: Chromatin immunoprecipitation (ChIP)]
Aliqoute the resuspended cells into Biorupotor Pico 1.5ml special tubes.
?. [Part #2: Chromatin immunoprecipitation (ChIP)]
Pour up to 1 mg protein (chromatin) in a 2-ml epi-tube as one replicate.
?. [Part #2: Chromatin immunoprecipitation (ChIP)]
Add Triton X100 from a 10% (v/v) stock to make the final conc. 1%Pause point: You may freeze the samples in -80°C for weeks.
?. [Part #3: Selective isolation of Chromatin-Asscotiated Proteins (SICAP)]
Put the tubes on the magnet, after 1min remove the liquid, and take the tubes off the magnet. Immidiately pour 1000ul of Tris-HCl 10mM pH 7.5 (without EDTA) on the beads. Process the tubes one by one. Never allow the beads to dry.Here the idea is to remove the inhibitors of the enzymatic reactions (e.g. EDTA). Stringent washes come later.
?. [Part #3: Selective isolation of Chromatin-Asscotiated Proteins (SICAP)]
Resuspend the beads by rotating end to end, and transfer them to a new 2ml epi-tube.
?. [Part #2: Chromatin immunoprecipitation (ChIP)]
Keep 1%, as the input control for ChIP-qPCR/seq
?. [Part #2: Chromatin immunoprecipitation (ChIP)]
Add proper amounts of your antibody to the samples.
?. [Part #2: Chromatin immunoprecipitation (ChIP)]
Agitate at 800RPM overnight in a Thermomixer (eppendorf) at 4°C or cold room
?. [Part #2: Chromatin immunoprecipitation (ChIP)]
After the overnight incubation, spin the samples at 12000g for 10min at 4°C
?. [Part #2: Chromatin immunoprecipitation (ChIP)]
Collect 95% of the liquid and transfer it into a new 2-ml epi-tubeYou may not see the pellet easily, however, sometimes there is a transparent precipitationA 2-ml epi-tube is necessary for efficient agitation in the next steps.
?. [Part #2: Chromatin immunoprecipitation (ChIP)]
Adjust the volume to 1ml by adding IP buffer.
?. [Part #2: Chromatin immunoprecipitation (ChIP)]
For each tube, you will need to prepare 40ul of ProteinA or ProteinG Dynabeads (depending on the antibody).
?. [Part #2: Chromatin immunoprecipitation (ChIP)]
Add 40ul magnetic Dynabeads to each tube
?. [Part #2: Chromatin immunoprecipitation (ChIP)]
Rotate the samples 2-4 hours at 4°C (cold room) on a rotating wheel.
?. [Part #3: Selective isolation of Chromatin-Asscotiated Proteins (SICAP)]
Resuspend the beads in 200ul of NEB Cutsmart buffer 1x. Don’t pipet the beads. Gently swirl the tube to resuspend the beads.
?. [Part #3: Selective isolation of Chromatin-Asscotiated Proteins (SICAP)]
Optional: If you wish to improve the efficiency of DNA end-labeling you may perform the folloing step. Although TdT adds nucleotides to 3’- and 5’-overhangs and blunt ends, Klenow 3’exo- adds biotinylated dATP to only 5’overhangs or blunt ends. Combination of these two enzymes improves DNA end-labelling about 10-20%. In addition, T4 PNK removes 3'-phosphates, and prepares the 3'-OH ends for TdT and Klenow.Prepare the following mixture for each tube. You may prepare a master mix for all the tubes:Cutsmart buffer 10x                 10 ulKlenow 3’exo-                             2 uldBTP (C+G+T) 10mM               1 ulBiotin-dATP 1mM                      10 ulT4 PNK                                        1ulFill up to 100ul with H2ONote: dBTP means al nucleotides except dATP.
?. [Part #3: Selective isolation of Chromatin-Asscotiated Proteins (SICAP)]
Put the samples one by one on the magnet, remove the liquid, and take the tubes off the magnet. Pour the mixture prepared in the previous step on the beads. Gently swirl the beads to resuspend the beads.
?. [Part #3: Selective isolation of Chromatin-Asscotiated Proteins (SICAP)]
Incubate at 37°C in a thermomixer with agitation at 700 RPM for 30 min.
?. [Part #3: Selective isolation of Chromatin-Asscotiated Proteins (SICAP)]
Put the samples one by one on the magnet, after 1min remove the liquid, and take the tubes off the magnet. Resuspend the beads in 100ul of TdT buffer 1x.
?. [Part #3: Selective isolation of Chromatin-Asscotiated Proteins (SICAP)]
Put the samples one by one on the magnet, remove the liquid, and take the tubes off the magnet. Resuspend the beads in the mixture prepared in step 55. Gently swirl the beads to resuspend the beads.
?. [Part #3: Selective isolation of Chromatin-Asscotiated Proteins (SICAP)]
To each tube add :TdT                                 4ulOptional: RNase A        1ulTdT is a quite specific enzyme for DNA labelling, as evident by TUNEL assay. Adding RNase A reassures specific DNA labelling.
?. [Part #3: Selective isolation of Chromatin-Asscotiated Proteins (SICAP)]
Incubate at 37°C in a thermomixer with agitation at 800 RPM for 30min.
?. [Part #3: Selective isolation of Chromatin-Asscotiated Proteins (SICAP)]
Wash the beads 4 times with 1ml ice-cold IP buffer at room temp.
?. [Part #3: Selective isolation of Chromatin-Asscotiated Proteins (SICAP)]
Remove the last IP wash buffer
?. [Part #3: Selective isolation of Chromatin-Asscotiated Proteins (SICAP)]
Resuspend the beads in 100ul, SDS 7.5% + DTT 200mM, and vortex vigorously
?. [Part #3: Selective isolation of Chromatin-Asscotiated Proteins (SICAP)]
Incubate 15min at 37°C in a thermomixer, with 1000 RPM
?. [Part #3: Selective isolation of Chromatin-Asscotiated Proteins (SICAP)]
Put the samples on the magnet, collect the liquid, and discard the beads
?. [Part #3: Selective isolation of Chromatin-Asscotiated Proteins (SICAP)]
Prepare the following mixture for each tube. You may prepare a master mix for all tubes:TdT buffer (including CoCl2)         20ulBiotin-dCTP                                        5ulBiotin-ddUTP                                      5ulFill up to 95ul with H2ONote: Biotin-dCTP was used to reduce the cost. You may use 10ul of biotin-ddUTP.
?. [Part #3: Selective isolation of Chromatin-Asscotiated Proteins (SICAP)]
Dilute the liquid with 1000ul IP buffer
?. [Part #3: Selective isolation of Chromatin-Asscotiated Proteins (SICAP)]
Add 70ul of Streptavidin magnetic beads to each tube. Rotate the tubes for 30-60min at room temptrature.
?. [Part #3: Selective isolation of Chromatin-Asscotiated Proteins (SICAP)]
Put the tubes on the magnet after 2 min discard the solution.
?. [Part #3: Selective isolation of Chromatin-Asscotiated Proteins (SICAP)]
Wash the beads 3 times by SDS wash bufferEach washing consists of resuspending the beads in the washing solution by vortexing, briefly spinning, and putting on the magnet for 2 min to remove the solutionAfter the second wash, normally you should be able to see the beads are dispersed, except the negative control.
?. [Part #3: Selective isolation of Chromatin-Asscotiated Proteins (SICAP)]
Wash with BW2x buffer, once
?. [Part #3: Selective isolation of Chromatin-Asscotiated Proteins (SICAP)]
Wash the beads with Iso-propanol wash buffer, once
?. [Part #3: Selective isolation of Chromatin-Asscotiated Proteins (SICAP)]
Wash the beads with acetonitrile wash buffer, twice
?. [Part #3: Selective isolation of Chromatin-Asscotiated Proteins (SICAP)]
Resuspend the beads in 80ul acetonitrile wash buffer, and transfer it into a PCR tubes.
?. [Part #3: Selective isolation of Chromatin-Asscotiated Proteins (SICAP)]
Put the tubes on the magnet, and remove the wash buffer.
?. [Part #3: Selective isolation of Chromatin-Asscotiated Proteins (SICAP)]
Spin the tubes briefly, and remove the residues of the wash buffer
?. [Part #4: Digesting proteins and sample preparation for mass spectrometry]
Resuspend the beads in 20ul Ambic 50mM + 10mM DTT (or TEAB 50mM for dimethylation or TMT labelling)Note: freshly prepared Ambic should be used.
?. [Part #4: Digesting proteins and sample preparation for mass spectrometry]
Incubate the beads at 50°C for 15min
?. [Part #4: Digesting proteins and sample preparation for mass spectrometry]
Chill the beads, and add 2ul of IAA 0.4MIAA: Iodoacetimide
?. [Part #4: Digesting proteins and sample preparation for mass spectrometry]
Vortex, and keep the tubes in a drawer for 15-30min
?. [Part #4: Digesting proteins and sample preparation for mass spectrometry]
Add  0.5ul of DTT 1M to neutralize IAA
?. [Part #4: Digesting proteins and sample preparation for mass spectrometry]
Add 300ng of LysC (3ul of 100ng/ul), vortex, and spin briefly
?. [Part #4: Digesting proteins and sample preparation for mass spectrometry]
Incubate overnight at 37 °C (12-16h)
?. [Part #4: Digesting proteins and sample preparation for mass spectrometry]
Remove the beads, and transfer the liquid into new PCR tubes.
?. [Part #4: Digesting proteins and sample preparation for mass spectrometry]
Heat the samples (without the beads) at 95 °C for 5min to reverse the formaldehyde crosslinking
?. [Part #4: Digesting proteins and sample preparation for mass spectrometry]
Chill the samples briefly
?. [Part #4: Digesting proteins and sample preparation for mass spectrometry]
Add 100ng of Trypsin (1ul of 100ng/ul)
?. [Part #4: Digesting proteins and sample preparation for mass spectrometry]
Incubate at 37°C for 8h.
?. [Part #4: Digesting proteins and sample preparation for mass spectrometry]
Add 1ul TFA 10% to the digested samples to acidify the pH
?. [Part #4: Digesting proteins and sample preparation for mass spectrometry]
Pour 50ul of Acetonitrile 80% + formic acid 0.1% in a glass insert (tubes for auto-sampler)
?. [Part #4: Digesting proteins and sample preparation for mass spectrometry]
Set a P20 on 20ul, and pick up a zip-tip
?. [Part #4: Digesting proteins and sample preparation for mass spectrometry]
Pipete 20ul Acetonitrile 100%, and discard the liquid. Repeat once again.
?. [Part #4: Digesting proteins and sample preparation for mass spectrometry]
Equilibrate the zip-tip by pipetting 20ul TFA 0.1%, and discard the liquid. Repeat twice again.
?. [Part #4: Digesting proteins and sample preparation for mass spectrometry]
Pipet one sample 10 times. Do not generate bubbles.
?. [Part #4: Digesting proteins and sample preparation for mass spectrometry]
Wash the zip-tip by pipetting 20ul TFA 0.1%, and discard the liquid. Repeat once again.
?. [Part #4: Digesting proteins and sample preparation for mass spectrometry]
Elute the peptides by pipetting Acetonitrile 80% + formic acid 0.1% in the glass insert
?. [Part #4: Digesting proteins and sample preparation for mass spectrometry]
Dry out the eluent in an appropriate speed vac.
?. [Part #4: Digesting proteins and sample preparation for mass spectrometry]
Reconstitute the peptides in 8ul DMSO 2% +  formic acid 0.1%
?. [Part #4: Digesting proteins and sample preparation for mass spectrometry]
Inject 7 out of 8 ul of the samples to the mass spec
?. [Appendix #2: Clean up the DNA by Ampure XP beads]
Following step 81, resuspend the beads in 23ul of SDS wash buffer
?. [Appendix #2: Clean up the DNA by Ampure XP beads]
Add 2ul of Proteinase K (20mg/ul)
?. [Appendix #2: Clean up the DNA by Ampure XP beads]
Incubate the beads at 55 °C for 15 min
?. [Appendix #2: Clean up the DNA by Ampure XP beads]
Incubate the beads at 95 °C for 10min
?. [Appendix #2: Clean up the DNA by Ampure XP beads]
Put the tubes on a magnetic rack, and remove the beads.
?. [Appendix #2: Clean up the DNA by Ampure XP beads]
Collect the liquid, and add 50ul of Ampure XP beads to the samples. Vortex, and wait 10min.
?. [Appendix #2: Clean up the DNA by Ampure XP beads]
Spin the samples, and put them on a magnetic rack to separate the beads.
?. [Appendix #2: Clean up the DNA by Ampure XP beads]
discard the liquid, and add 200ul of freshly prepared EtOH 80%, and wait a few second
?. [Appendix #2: Clean up the DNA by Ampure XP beads]
10-discard the liquid, repeat the previous step once again.
?. [Appendix #2: Clean up the DNA by Ampure XP beads]
Spin the samples, and put them on a magnetic rack to separate the beads.
?. [Appendix #2: Clean up the DNA by Ampure XP beads]
Discard the residual ethanol.
?. [Appendix #2: Clean up the DNA by Ampure XP beads]
Resuspend the beads in 20ul of Tris-Cl 10mM pH 8.0
?. [Appendix #2: Clean up the DNA by Ampure XP beads]
Spin the samples, and put them on a magnetic rack to separate the beads.
?. [Appendix #2: Clean up the DNA by Ampure XP beads]
Collect the samples, and transfer them to new tubes. The samples can be used for qPCR or library preparation.
?. [Appendix #1: Modification of streptavidin beads to avoid streptavidin contamination]
Pour 5ml of streptavidin beads (e.g. NEB S1420S) into a 15mL tube
?. [Appendix #1: Modification of streptavidin beads to avoid streptavidin contamination]
Transfer the tube on the magnet, wait 5min and then discard the liquid
?. [Appendix #1: Modification of streptavidin beads to avoid streptavidin contamination]
Wash the beads once again with 10mL of PBS-TPBS-T : PBS + Tween20 0.1% (v/v)
?. [Appendix #1: Modification of streptavidin beads to avoid streptavidin contamination]
Put the tube on the magnet, wait 5min and then discard the liquid
?. [Appendix #1: Modification of streptavidin beads to avoid streptavidin contamination]
Resuspend the beads in 7ml of 0.2M Sodium cyanoborohydride in PBS-T.
?. [Appendix #1: Modification of streptavidin beads to avoid streptavidin contamination]
Add 7ml of Formaldehyde 4%
?. [Appendix #1: Modification of streptavidin beads to avoid streptavidin contamination]
Rotate 2 hours at room temperature
?. [Appendix #1: Modification of streptavidin beads to avoid streptavidin contamination]
Put the tube on the magnet, wait 5min and then discard the liquid
?. [Appendix #1: Modification of streptavidin beads to avoid streptavidin contamination]
Wash the beads with 10ml Tris.Cl 0.1M pH 7.5
?. [Appendix #1: Modification of streptavidin beads to avoid streptavidin contamination]
Put the tube on the magnet, wait 5min and then discard the liquid
?. [Appendix #1: Modification of streptavidin beads to avoid streptavidin contamination]
Wash the beads with 10ml PBS-T
?. [Appendix #1: Modification of streptavidin beads to avoid streptavidin contamination]
Put the tube on the magnet, wait 5min and then discard the liquid
?. [Appendix #1: Modification of streptavidin beads to avoid streptavidin contamination]
Resuspend the beads in 5mL PBS-T
?. [Appendix #1: Modification of streptavidin beads to avoid streptavidin contamination]
14-Keep the beads in the fridge. The beads are stable at +4⁰C for months