Modified from Illumina's Protocol (Part # 11251892 Rev. A)
1) In a 0.6ml thin-walled PCR tube, add 10μl ChIP DNA.
2) Dilute just enough Klenow Enzyme (DNA polymerase) 1:5 with dH20 for a final Klenow concentration of 1U/μl.
3) Make Master Mix for the following:
End Repair |
Vol |
x |
|
dH20 |
30μl |
|
|
T4 DNA ligase buffer with 10mM ATP |
5μl |
|
|
dNTP mix |
2μl |
|
|
T4 DNA Polymerase |
1μl |
|
|
Klenow DNA Polymerase (1/5 dilution) |
1μl |
|
|
T4 PNK |
1μl |
|
|
Total: |
40 |
|
4) Add 40μl of End Repair mix to each DNA sample for a total volume of 50μl.
5) Incubate in thermal cycler for 30 min at 20°C, hold samples on ice.
6) Purify samples with QIAquick PCR Purification Kit.
* Add 250μl of Buffer PBI to sample and mix by pipetting.
* Check that the color of the mixture is yellow (if not see kit directions).
* Transfer sample to QIAquick column fitted into a 2ml collection tube.
* Centrifuge for 60 sec at 16000xg. Discard flow-through and put column back into the same tube.
* To wash, add 750μl Buffer PE (make sure EtOH has been added).
* Centrifuge for 60 sec at 16000xg. Discard flow-through and put column back into the same tube.
* Centrifuge for an additional 60 sec at 16000xg with caps open.
* Transfer column to clean 1.7ml microcentrifuge tube.
* Apply 34μl Buffer EB to column and incubate 1min at room temp.
* Centrifuge for 60 sec at 16000xg.
7) Measure the volume of the samples and q.s. to 34μl with water. Samples can be stored at -20°C.
8) Prepare the following reaction mix:
|
A' Base Addition |
Vol |
x |
|
dATP 1mM |
10μl |
|
|
Klenow Buffer |
5μl |
|
|
Klenow Exo |
1μl |
|
|
Total: |
16 |
|
9) Add the 16μl of A' Base Addition mix to each tube containing 34μl of DNA.
10)Incubate in a 37°C waterbath for 30 min.
11)Purify samples with MinElute PCR Purification Kit.
* Add 250μl of Buffer PBI to sample and mix by pipetting.
* Check that the color of the mixture is yellow (if not see kit directions).
* Transfer sample to MinElute column (stored at 4°C).
* Centrifuge for 60 sec at 16000xg. Discard flow-through and put column back into the same tube.
* To wash, add 750μl Buffer PE (make sure EtOH has been added).
* Centrifuge for 60 sec at 16000xg. Discard flow-through and put column back into the same tube.
* Centrifuge for an additional 60 sec at 16000xg with caps open.
* Transfer column to clean 1.7ml microcentrifuge tube.
* Apply 10μl Buffer EB to column and incubate 1min at room temp.
* Centrifuge for 60 sec at 16000xg. Samples can be stored at -20°C.
12)Dilute just enough Oligo mix 1:10 with dH20.
13) Prepare the following reaction mix:
|
Ligation |
Vol |
x |
|
DNA Ligase Buffer |
15μl |
|
|
Oligo Mix (1/10 dilution) |
1μl |
|
|
DNA Ligase |
4μl |
|
|
Total: |
20 |
|
14)Add 20μl of Ligation mix to each tube containing 10μl of DNA sample.
15)Incubate for exactly 15 min at room temperature, hold samples on ice.
16)Purify samples with MinElute PCR Purification Kit.
* Add 150μl of Buffer PBI to sample and mix by pipetting.
* Check that the color of the mixture is yellow (if not see kit directions).
* Transfer sample to MinElute column (stored at 4°C).
* Centrifuge for 60 sec at 16000xg. Discard flow-through and put column back into the same tube.
* To wash, add 750μl Buffer PE (make sure EtOH has been added).
* Centrifuge for 60 sec at 16000xg. Discard flow-through and put column back into the same tube.
* Centrifuge for an additional 60 sec at 16000xg with caps open.
* Transfer column to clean 1.7ml microcentrifuge tube.
* Apply 10μl Buffer EB to column and incubate 1min at room temp.
* Centrifuge for 60 sec at 16000xg. Samples can be stored at -20°C.
17)Prepare a 200ml 2% agarose 1X TAE gel in the large gel tray. The gel can be pre-stained with SyBR-Safe (10μl per 100ml of gel). Put fresh running buffer in the gel tank plus 20μl SyBR-Safe at the 'red' end every time – this is very important to prevent contamination from old buffer and to keep dye from running off.
18)Add 3μl of loading buffer (50mM Tris pH 8.0, 40mM EDTA, 40%w/v sucrose) to each purified, ligated, DNA sample (13μl total).
19)Load 5μl of Ccl9 and Cxcl11 PCR products in first two lanes. Load 4μl of 50bp ladder (SM0373 Fermentas) to every 4th lane.
20)Skipping lanes between samples and ladders, load the entire DNA sample into a well. Also have a blank well for the control (with empty lanes on either side)
21)Run gel: 70V for 1hr, 80V for 2hrs, 100V for 3hrs.
22)Take a picture of the gel.
23)Excise samples (180bp-300bp) using a clean razor blade for each sample, and place into a 2.0ml tube. Also cut out the gel from the control lane. Take a picture of the gel to show regions that were cut out.
24)Weigh samples and record weight. Gel slices can be stored at -20°C. Gel slices >0.4g must be treated differently (see kit).
25)Use a QiaQuick Gel Extraction Kit to purify the DNA from the agarose.
* Add 300μl of Buffer QG for each 100mg of gel.
* Incubate in 55°C waterbath for 10-30min until gel is completely dissolved.
* Check that the color of the mixture is yellow (if not see kit directions).
* Add 1 gel volume of Isopropanol and mix by pipetting. Do not centrifuge.
* Transfer sample to QIAquick column (max 750μl) fitted into a 2ml collection tube.
* Centrifuge for 60 sec at 16000xg. Discard flow-through and put column back into the same tube. If there is additional sample left, reload and re-spin.
* Add 500μl Buffer QG to column.
* Centrifuge for 60 sec at 16000xg. Discard flow-through and put column back into the same tube.
* To wash, add 750μl Buffer PE (make sure EtOH has been added).
* Centrifuge for 60 sec at 16000xg. Discard flow-through and put column back into the same tube.
* Centrifuge for an additional 60 sec at 16000xg with caps open.
* Transfer column to clean 1.7ml microcentrifuge tube.
* Apply 36μl Buffer EB to column and incubate 1min at room temp.
* Centrifuge for 60 sec at 16000xg.
26) Measure the volume of the samples and q.s. to 36μl with water. Samples can be stored at -20°C.
27)Prepare reaction mix (make enough for 1.5 extra samples as this mix is very bubbly):
|
Enrichment |
Vol |
x |
|
5X Phusion Buffer |
10μl |
|
|
dNTP mix |
1.5μl |
|
|
PCR Primer 1.1 |
1μl |
|
|
PCR Primer 2.1 |
1μl |
|
|
Phusion Polymerase |
0.5μl |
|
|
Total: |
14 |
|
28)Add 14μl of Enrichment mix to a 0.6ml thin-walled PCR tube.
29)Add 36μl of DNA.
30)Amplify using following protocol:
98°C for 30 sec
18 cycles of:
98°C for 10 sec
65°C for 30 sec
72°C for 30 sec
72°C for 5 min
4°C hold
31)Purify samples with MinElute PCR Purification Kit.
* Add 250μl of Buffer PBI to sample and mix by pipetting.
* Check that the color of the mixture is yellow (if not see kit directions).
* Transfer sample to MinElute column (stored at 4°C) fitted into a 2ml collection tube.
* Centrifuge for 60sec at 16000xg. Discard flow-through and put column back into the same tube.
* To wash, add 750μl Buffer PE (make sure EtOH has been added).
* Centrifuge for 60sec at 16000xg. Discard flow-through and put column back into the same tube.
* Centrifuge for an additional 60 sec at 16000xg with caps open.
* Transfer column to clean 1.7ml microcentrifuge tube.
* Apply 15μl Buffer EB to column and incubate 1min at room temp.
* Centrifuge for 60sec at 16000xg. Samples can be stored at -20°C.
Have Samples BioAnalyzed on DNA chipsThe 36-bp reads are aligned to the reference mouse genome (mm9) using the ELAND program in "extended" mode (see the Sequencing Analysis Software User Guide from Illumina). Reads are aligned with a seed length of 25 bp, and the full-sequence alignment score is computed for the 15 best candidate matches based on the seed length, to select the best alignment. Reads that align to a single location in the genome (hereafter called "tags") are then extended in the 3' direction using the reference genome, to a fragment length of 250 bp. For each combination of genome location and strand, at most a single fragment matching that location and strand is retained (all other fragments matching that location and strand are excluded on the basis of being likely PCR-derived copies of a single fragment from the immunoprecipitation); each such allowed fragment will be called an extended fragment. At survey points established every 10 bp along the chromosome, the number of overlapping extended fragments is counted. The survey point counts are then rescaled into "tags per million" (TPM) by dividing by the total number of extended fragments and multiplying by one million. Binding locations are detected by comparing the target-specific IP to a replicate-averaged negative control IP ("background") at each survey point in the genome. To identify a specific location as belonging to a binding location ("peak"), three conditions must hold: (1) the replicate-averaged background value (in TPM) at that location must not exceed 0.7; (2) the ratio of the target-specific IP value must exceed the background value by at least five-fold; and (3) the target-specific IP value must exceed the greater of 8 fragments or 1.5 TPM, at that location. If a single survey point satisfies all of the above criteria, it is flagged as a binding event, and the span of all survey points within plus or minus 200 bp satisfying the criteria is defined as the peak region. The data processing is carried out using custom software written in MATLAB.