Wednesday, June 22, 2011

Chromatine immuno-precipitation (ChIP) - lab protocol


This is the protocol for chromatin immuno-precipitation or ChIP. It was optimized in 2007 during my PhD work in the lab of Prof. Nicolas Mermod at University of Lausanne. In my experience it may be better to buy a kit as it is already optimized, but if your lab has limited money and experiment is complex and would require a lot kits/money spent, and you have time to spend on optimization (it is fun though) try this protocol.

The Protocol works very well with antibodies against various histone modifications, as exemplified in the figure bellow. When promoter of the housekeeping gene GAPDH was tested in mouse embryonic fibroblasts, histone H3 and H4 acetylations (markers of euchromatin) showed ~10 fold increased signal over the controls with no antibody or with the GAPDH antibody. This happened in both cell types that we tested, wild-type fibroblasts and knock-out fibroblasts for Nuclear Factor I transcription factor. In the same time histone modifications for heterochromatin did not show an increase and were at the level of no antibody controls, as expected since the GAPDH gene is highly expressed.

Note 1: this protocol is actually extended to fit the need of doing parallel experiments in the lab. It can be shortened at several overnight steps (by replacing them with 2 hour incubation) so that it can be all together finished in 5 or so days. However, if you are doing in parallel other experiments I highly recommend following this protocol as it will leave you few days almost free to do other things in the lab.

Note 2: In paper that we published using this protocol, doi:10.1186/1471-2164-12-181,  enrichment for transcription factor was relatively weak. However we managed to extract peaks and show global trends in the data, but due to low enrichment we had an issue of large number of false positives. It might be that the antibody was poor, but also might be that the this protocol is not suitable for TFs for currently unknown reason. Thus, be careful if you are using it for TFs. Test by qPCR before NGS sequencing.




Chromatin immunoprecipitation experiment with cultured mouse
embryonic fibroblasts (MEF):

- Mouse embryonic fibroblasts were extracted from embryos of 14.5 days
- Cell from embryos were cultured in DMEM medium under the following conditions:
  37°C
  5% CO2
  DMEM (GIBCO, 41966),
  Supplementary 10% FBS (GIBCO, Fetal Bovine Serum, qualified origin US, 26140-079)
  1% v/v nonessential amino-acids (GIBCO, 11140-035)
  1% v/v L-glutamine (GIBCO, 25030-024)

- Chromatin was extracted from the cells and the IP was performed using the following protocol and the commercial antibody against NFI group of proteins (NFI (H300): sc-5567, SantaCruz Biotechnology)

ChIP protocol

Day 1
1. Fixing of cells
- Approximately 20.000.000 cells from a culture (4 – 5 big plates)
- Add 0.1 volume of chromatin fixation buffer

Chromatin fixation buffer, Stock, Volume in 50ml
11% formaldehyde, 37%, 15 ml
0.1M NaCl, 5M, 1 ml
50 mM HEPES ph 7.9, 1M, 2.5 ml

- Incubate 7’ on RT with gentle shaking
- stop the fixation by adding 0.1 volume of 2M Glycine (pH not adjusted), color turns to yellow
- Incubate 5’ on RT with gentle shaking
- Wash 1x with ice cold PBS with protease inhibitors
- Scrape cells in ice cold PBS with protease inhibitors
- Transfer in 50 ml tubes
- Spin down in cold cetrifuge, 5’, 2500rpm
- Throw away the supernatant
2. Isolation of the chromatin
- Resuspend pellet in 5ml ice cold Cell-Lysis buffer, use blue tip to resuspend the pellet
- 10’ on ice
- Pellet nuclei and cell debris, cold centrifuge, 5’, 2500rpm
- Discard supernatant
- Resuspend pellet in 5ml Nuclear-Lysis buffer, use blue 1ml tips
- 5 min on ice
- Spin down, cold centrifuge, 5min, 2500rpm
- Resuspend in 2ml of ice cold PBS with protease inhibitors
- Spin down, cold centrifuge, 5’, 2500rpm
3. Chromatin sonication
- Resuspend chromatin in 1.5ml TEN buffer
- Transfer to 5ml conical tube (avoid polystyrene tubes since they can break)
- Sonicate 20x 10sec; On VibraCell-75455 (Bioblock Scientific) set Amplitude - 30, Pulser - 3 sec, if you put 40 & 4 it will most likely start foaming
- Cool down between cycles in ethanol/dry-ice bath
- Avoid foaming, do not move the tube to the top – foaming, or to the bottom – tube will break
- Transfer samples to eppendorf tubes
- Spin down 15min 14000rpm, cold centrifuge
- Save supernatant (chromatin prep)
- Add NaDOC to 0.1% final concentration (without NaDOC clumps will precipitate after freeze/thaw)

Day 2
4. Reverse cross-link

- Take 50ul or 100ul of the chromatin
- Add 500ul of Elution buffer
- Incubate 10’ at 65°C
- Add 11ul 5M NaCl
- Add 7ul Proteinase K (20mg/ml)
- Incubate 2h at 42°C
- Incubate O/N at 65°C

Day 3
5. Purification of the DNA (Phenol-Chloroform)
- Add equal volume of Phenol ~ 600ul
- Shake, vortex a little bit
- Spin 2 min, 12000 rpm, 4°C
- Transfer supernatant to a fresh tube (avoid aspiration of the interlayer or organic phase)
- Add equal volume of Chloroform /Isoamilalcohol (24:1) ~ 600ul
- Shake, vortex a little bit
- Spin 2 min, 12000 rpm, 4°C
- Transfer supernatant to a fresh tube (avoid aspiration of the interlayer or organic phase)
- Add 0.1 volume of 3M sodium acetate (~ 60ul)
- Add 1ul of glycogen (20 ug/ul),
- Add 1ml of isopropanol, don’t add glycogen after isopropanol it will make clumps
- Not necessary to be in cold but put 1h, -20°C, don’t put in -80°C it will freeze
- Spin 20’, 14000, 4°C
- Carefully take the supernatant with the blue tip, don’t lose the pellet
- Carefully add 1ml cold 70% Ethanol (don’t vortex)
- Spin 10 min, 14000, 4°C
- Carefully take the supernatant with the blue tip and what is left take with the yellow tip, don’t lose the pellet
- Air dry in the hood for 10-15 min (do not overdry because DNA becomes hard to
dissolve)
- Dissolve in 50 or 100 ul of water
- Keep on -20°C because unbuffered DNA may undergo degradation

Day 4
6. Check the average fragment length and concentration

- Add 5ul RNaseA, 1h 37°C
- Put 5ul of DNA on agarose gel (1%)
- Check the fragment size, should be between 100-1000bp, with an average at 500bp
- Take 10ul, add 40ul of H2O (5x dilution)
- Check the concentration of the DNA on spectrophotometer, conc=A260 x 5 x 50ng/ul, A260 should be > 0.2
- If necessary make 10x dilution, if A260 > 2
- Concentration should be around 250ng/ul
Gel pictures of sonicated chromatin:

DNA fragments size average at 1000bp, needs additional sonication

DNA fragments size average bellow 500bp, well sonicated chromatin

Day 5
7. IP reaction

- Take N x 10ul of Protein A Sepharose beads, N – number of IP reactions
- Wash the beads 2x with IPB
- Washing cycle: add 1ml of the solution
- Tumble on the rotator for 3 min
- Spin down 1’ 3000 rpm
- Wash the beads with dilution-incubation buffer, to saturate the beads with salmon
DNA, so they would not bind you’re complexes when preclearing the chr, but just some other components present in the solution that could interfere with IP
- Defrost chromatine
- Take 10ug of chromatin per reaction, take 50ug per reaction if you plan to do IP for transcription factors
- Dilute chromatin in 300ul of dilution-incubation buffer – N x 300ul of D-I buffer + N x 10ug
of chromatin, if necessary use 15ml yellow tubes. Why are we mixing our chromatin with salmon sperm DNA? Avoid this step and use some other buffer
- we use TEN buffer in a ratio 1:1 in order to put everything in a 1.5ml tube; but possibly dilute in a ratio 1:3 and divide in multiple 1.5ml tubes containing Nx10/M ul of beads, M - number of tubes, should be calculated from the amount of chr used; (p+3p)/1.5, p – ml of chr picked
- Suspend beads in chromatin dilution
- Incubate 30’ with tumbling, cold room
- Spin down 1’ 3000 rpm
- Keep the supernatant, don’t take any beads
- Spin at 14000 rpm 15’
- Take the supernatant
- Dispense supernatant containing 10 ug of chr into safelock ependorf tube with 700 ul of DI buffer; again why are we mixing our DNA with salmon’s DNA, use other buffer.
- we use 700ul of TEN buffer
- Keep the 1x supernatant with 10ug of chr as an input - only don’t mix with 700ul of DI
- Defrost antibodies
- Add directly to precleared chromatin, 2ug per 10ug of chr
- Incubate overnight, cold room, with slow tumbling

Day 6
8. Clearing the IP reactions with the beads

- Take Protein A Sepharose beads, N x 10ul
- Wash them 2x with IPB
- Washing cycle: add 1ml of the solution
- Tumble on the rotator for 3 min
- Spin down 1’ 3000 rpm
- Wash the beads 1x with DI buffer this time to reduce the background (unspecific binding to the DNA – so beads pick up DNA-histone complexes that are not flagged by Ab), do not centifuge
- Mix to avoid sedimentation, divide the beads equally into N tubes, N – number of IP
- Centrifuge IP reactions 10’ 10krpm, 4°C, take supernatant, avoid this step!
- Put supernatant onto the beads
- Mix
- Incubate O/N, cold room

Day 7
9. Washing the beads and Eluting the DNA from the beads

- let the beads sediment for 5 min
- centrifuge 3000rpm, 1min
- take all the SN
- perform N washes with IPB
- Washing cycle: add 1ml of the solution
- Tumble on the rotator for 3 min
- Spin down 1’ 3000 rpm
- Add 500ul of elution buffer to the beads
- Incubate ON with slow tumbling on the rotator, RT

Day 8
- centrifuge 3000rpm, 1min
- take all the SN
- Make the input, add 500ul of elution buffer to the input chr
- Incubate 10 min on 65°
- Add 11ul 5M NaCl
- Add 7ul Proteinase K (20mg/ml)
- Incubate 2h at 42°C
- Incubate O/N at 65°C

Day 9
10. Phenol-Chloroform DNA purification
- Add equal volume of Phenol ~ 600ul
- Shake, vortex a little bit
- Spin 2 min, 12000 rpm, 4°C
- Transfer supernatant to a fresh tube (avoid aspiration of the interlayer or organic phase)
- Add equal volume of Chloroform /Isoamilalcohol (24:1) ~ 600ul
- Shake, vortex a little bit
- Spin 2 min, 12000 rpm, 4°C
- Transfer supernatant to a fresh tube (avoid aspiration of the interlayer or organic phase)
- Add 0.1 volume of 3M sodium acetate (~ 60ul)
- Add 1ul of glycogen (20 ug/ul),
- Add 1ml of isopropanol, don’t add glycogen after isopropanol it will make clumps
- Not necessary to be in cold but put 1h, -20°C, don’t put in -80°C it will freeze
- Spin 20’, 14000, 4°C
- Carefully take the supernatant with the blue tip, don’t lose the pellet
- Carefully add 1ml cold 70% Ethanol (don’t vortex)
- Spin 10 min, 14000, 4°C
- Carefully take the supernatant with the blue tip and what is left take with the yellow tip, don’t lose the pellet
- Air dry in the hood for 10-15 min (do not overdry because DNA becomes hard to dissolve)
- Dissolve in 50 or 100 ul of water
- Keep on -20°C because unbuffered DNA may undergo degradation

Tuesday, June 21, 2011

Paper: "High-Resolution Analysis of Parent-of-Origin Allelic Expression in the Mouse Brain", extracts

Science 6 August 2010:
vol. 329 no. 5992 pp. 643-648

High-Resolution Analysis of Parent-of-Origin Allelic Expression in the Mouse Brain
Christopher Gregg, Jiangwen Zhang, Brandon Weissbourd, Shujun Luo, Gary P. Schroth, David Haig, Catherine Dulac

This paper shows that in mice there are 8 times more genes that are imprinted than it was previously known and that imprinting is more of a continuum of differential expression between parental alleles than strict monoallelic expression as previously defined.

Extracts:

-Currently, fewer than 100 imprinted genes have been identified.

-Parent-specific biases emerged as a continuum from the data set, which suggested that imprinting may manifest as relative allele-specific expression bias, rather than strict monoallelic transcription, or that allelic bias is cell-type specific and is partially masked by cellular heterogeneity in brain samples.

-Imprinted genes and genes with imprinted features were identified by the presence of one or more SNP sites exhibiting a significant paternal or maternal expression bias, as described above. This approach enabled us to identify 1308 candidate imprinted loci, among which were 824 genes annotated in the University of California Santa Cruz genome database (UCSC) and 484 putative noncoding RNAs (ncRNAs) annotated in the functional RNA database (fRNAdb) (4.1% of the 11,545 ncRNAs assessed).

-Sixty-one percent of genes identified in the E15 brain were maternally expressed genes (MEGs), which revealed a significant maternal bias in the developing brain [paternally expressed genes (PEGs), 215; MEGs, 338; P < 0.0001; hi2 analysis). In contrast, a paternal bias was observed in both the adult pre optic area POA (PEGs, 172; MEGs, 84; P < 0.0001; hi2 analysis) and the adult medial prefrontal cortex mPFC (PEGs, 109; MEGs, 44; P < 0.0001; hi2 analysis), such that ~70% of genes identified in the adult brain were PEGs.