Linear Amplification and labelling of ChIP fractions for hybridization on Affymetrix arrays

this is Version 1.2

You’re supposed to have Immunoprecipitated chromatin, purified DNA from this ChIP,  as described in Liu et al. PLoS Biol 2005 3(10):1753-1769. Your DNA is now in 20μl H2O. You need to know the concentration of it, which is measurable with OliGreen labelling and a standard curve, as described here .

 

This protocol will then amplify (as in Liu et al. BMC Genomics 2003, 4 :19) your ChIP for Affymetrix microarray hybridization. We are grateful to Chih Long Liu for providing detailed guidelines of his protocol on the TLAD web page .

Note : Before beginning this protocol, if your source DNA was MNase-treated, you may need to CIP-treat the samples to remove 3’ phosphate groups.

Optional: CIP Treatment of samples

For 20µL sample : 0,75µL CIP+ 30µL NEB3 1X
Incubate at 37°C for 1 hour.
Clean up reaction with the Qiagen MinElute kit. Elute in 20µL.
Concentrate in SpeedVac to a final volume of 6μl.

Step 1 : Poly-T tailing

Prepare in PCR tubes :

ChIP DNA:                                     6 μl
Roche TdT 5X reaction buffer:        2 μl        (1X final)
Roche CoCl2 (25mM):                 0.5 μl        (0.75mM final)
8% 100µM ddCTP in 100µM dTTP: 0.5 μl        (5 μMdTTP  with 8% ddCTP final)
20U TdT Transferase (NEB):            1 μl        (2U/µL final).

Notes:
*Ensure that the dNTP mixes do not go through more than 3-freeze-thaw cycles.
Additionnal freeze-thaw cycles will further degrade the dNTPs and will reduce the efficiency of the reaction.
*It is strongly suggested that the NEB enzyme is use for this protocol. TdT enzyme from other sources may not perform optimally.
*Do not use the NEB Buffer 4 supplied with the NEB enzyme, since the DTT in the buffer will precipitate the CoCl2 and inhibit the reaction. Use the Cacodylate buffer supplied with the Roche enzyme.

Incubate 20 minutes at 37°C in a thermocycler.
Halt by  adding 2μl of EDTA 0.5 M ph8.0
Purify on Qiagen MinElute Reaction Cleanup kit (add 10µL H2O before the reaction clean up to bring the volume up to 20µL), elute in 20μl H2O Millipore.
Concentrate in SpeedVac to a final volume of 6.5μl.

Step 2: Second-strand synthesis, T7 promoter fusion

Wear gloves and work in the “RNase free area”

Prepare in PCR tubes :

DNA eluate:	6.5 μl
T7-A18B primer (1 μM):	0.75 μl
NEB 2 Buffer:	1 μl
dNTPs at 5 mM (each):	0.4 μl
H2O:	0.95µl

Apply following cycling:
94°C for 2 min
ramp down -1°C/sec to 35°C
35°C for 2 min
ramp down -0.5°C/sec to 25°C, hold at 25°C and add 0.4 μl of Klenow (2U).
Incubate at 37°C for 90 minutes.
Halt by adding 1.25 μl of EDTA 0.5M pH8.0
Clean on MinElute Reaction Cleanup QIAgen kit (dedicated to RNA-quality work), elute in 20 μl H20 RNase-free.
Concentrate in SpeedVac to a final volume of 5μl.

Step 3: IVT-Labelling

Wear gloves and work in the “RNase free area”

Note: We observed a twice-higher efficiency with Ambion MEGAShortscript kit as compared to the standard MEGAscript kit

Using the Ambion  MEGAshortscript Kit and the NTP Mix from the "T7 IVT labeling kit" of Affymetrix:

H2O (Nuclease free):                                    5µL
IVT Labeling NTP mix (Affymetrix):                6µL
Ambion T7 10X reaction buffer (warm to RT): 2µL
Template DNA:                                             5µL
Ambion T7 Enzyme mix:                               2µL

Incubate at 37°C overnight, for 16h, preferentially in a PCR thermocycler equipped with heated lid to minimize vapour volumes

Step 4: RNA Purification

Use the Qiagen RNeasy Mini kit, quantitate your RNA on Nanodrop, proceed to microarray hybridization if at least 15 micrograms were produced.

Note for the elution: Eluate with 50µL H2O, then eluate again with the same 50µL.

Buffers, Reagent list:

ddCTP (10mM, ref K043.1 from Roth): dilute to 100 μM in H2O before use.

dTTP (100mM, ref T9656 from Sigma): dilute to 100 μM in H2O before use.

TdT 5X reaction buffer and CoCl2  ( ref 03 333 566 001; Roche)

TdT Transferase (NEB) (ref M0252S; Ozyme)

QIAgen MinElute Reaction Cleanup kit

QIAgen RNeasy Mini kit

MEGAshortscript Kit (ref AM1354; Ambion; Applied Biosystems)

Dernière mise à jour : ( 19-03-2010 )