ChIP sequencing and analysis

1. Dissect gonads and fix them in 1% PFA for 10 min at room temperature, add, at least, 10 times the volume of the tissue.

2. Stop fixation by adding 1/10 the volume of the fixation of Glycine 1.25M.

3. Wash 2 times with cold 1X PBS.

4. Remove all the PBS and snap freeze the tissue.

5. Store at -80°C or continue to the next step using the iDeal ChIP-seq kit for Histones (Diagenode, Cat. No. C01010051) or the the iDeal ChIP-seq kit for Transcription Factors (Cat. No. C01010054).

·      iDeal ChIP-seq kit for Histones (Diagenode, Cat. No. C01010051)

1.  Add 10 ml of cold Lysis buffer iL1 to the tissue and incubate for 10 min at 4°C with gentle mixing.

2. Centrifuge for 5 min at 500 x g at 4°C and discard the supernatant.

3. Add 10 ml of cold Lysis buffer iL2 to the tissue and incubate for 10 min at 4°C with gentle mixing.

4. Centrifuge for 5 min at 500 x g at 4°C and discard the supernatant.

5. Add 1ml of the Shearing buffer iS1 complemented with 200x protease inhibitor cocktail.

6. Shear the chromatin in aliquots of 300 µl in low binding tubes (recommended: Bioruptor® Plus 1.5 ml TPX Microtubes (Cat. No. C30010010). Use High power setting for 45 cycles (30 seconds ON, 30 seconds OFF). Stop the system after each run of 10 cycles, vortex and spin down sample.

7. Centrifuge at 16,000 x g for 10 min at 4°C and collect the supernatant which contains chromatin.

8. Take 10 µl of chromatin for de-crosslinking and concentration estimation. Snap freeze the rest and store at -80°C

            De-Crosslinking:

                        8.1. Add 1 µl of 5M NaCl and incubate O/N at 65°C.

                        8.2. Add 1 µl of RNAse 10mg/ml and incubate 1h at 37°C.

                        8.3. Add 1 µl of PK 20mg/ml and incubate 1h at 56°C.

                        8.4. Precipate the DNA by adding 1/10 of NaOAc and 3 times of EtOh 100%. Centrifuge at 16,000 x g for 30 min at 4°C and discard the supernatant.

                        8.5. Wash the pellet with 1ml of EtHO 70%. Centrifuge at 16,000 x g for 10 min at 4°C and discard the supernatant.

                        8.6. Dry the pellet for 15min at room temperature, resuspend the pellet in 10 µl of H2O and measure the DNA concentration with nanodrop or cubit and estimate the efficiency of shearing by loading it in 1% agarose gel.

9. Wash 4 times the DiaMag Protein A-coated magnetic beads (20 µl/immunoprecipitation, IP) with twice the volume of ice-cold 1x ChIP buffer iC1. Resuspend the beads in 1 volume of 1x ChIP buffer iC1. Set aside 1 µl of the sheared chromatin to use as input sample and keep at 4°C.

10. Prepare the following ChIP reaction mix (1 IP):

• 6 µl of 5% BSA

• 1.5 µl of 200x protease inhibitor cocktail

• 56 µl of 5x ChIP buffer iC1

• x µl of sheared chromatin (typically for Histones 5 µg per IP).

• 20 µl of DiaMag Protein A-coated magnetic beads

• 1 µg ChIP-seq antibody

• add ChIP-seq grade water to a total volume of 300 µl

11. Incubate overnight at 4°C on a rotating wheel.

12. The next day, briefly spin the tubes, place them in the ice-cold magnetic rack and discard the supernatant.

13. Add 350 µl ice-cold Wash buffer iW1 and incubate for 5 min at 4°C on a rotating wheel. Discard the wash buffer using the Diagenode magnetic rack.

14. Repeat the previous step once with Wash buffer iW2, iW3 and iW4, respectively.

15. After removing the last wash buffer, add 100 µl of Elution buffer iE1 to the beads and incubate for 30 min on a rotating wheel at room temperature. Resuspend the beads pellet and transfer it into a new 200 µl tube.

16. Briefly spin the tubes and place them into the Diagenode magnetic rack. Transfer the supernatant to a new tube and add 4 µl of Elution buffer iE2. Also add 99 µl buffer iE1 and 4 µl of buffer iE2 to the 1 µl input sample. Incubate for 4 hours or overnight at 65°C.

17. Purify the DNA using by adding 2 µl of carrier to each IP and input sample. 5

18. Add 100 µl of 100% isopropanol to each IP and input sample.

19. Resuspend the IPure beads v2 and transfer 10 µl to each IP and input sample.

20. Vortex well and incubate 10 minutes at room temperature on a rotating wheel.

21. Prepare the Wash buffer 1 containing 50% isopropanol.

22. Briefly spin the tubes, place into the DiaMag0.2 - magnetic rack, wait 1 min and discard the buffer. Add 100 µl Wash buffer 1 in each tube. Close the tubes, incubate for 30 seconds at room temperature on a rotating wheel.

23. Prepare the Wash buffer 2 containing 50% isopropanol.

24. Briefly spin the tubes, place into the DiaMag0.2 - magnetic rack, wait 1 min and discard the buffer. Keep the captured beads and add 100 µl Wash buffer 2 per tube. Close the tubes, resuspend the beads and incubate for 5 min at room temperature on a rotating wheel.

25. Briefly spin the tubes and place them into the DiaMag0.2 or DiaMag1.5, wait 1 minute and discard the buffer. Keep the captured beads and add per tube 25 µl buffer C. Close the tubes, invert the 8-tube strip to resuspend the beads and incubate for 15 minutes at room temperature on a rotating wheel. Resuspend the pelleted beads by pipetting.

26. Spin the tubes and place them into the DiaMag0.2 - magnetic rack, wait 1 min and transfer the supernatants into a new labelled 1.5 ml tube and discard the beads.

27. Proceed to library preparation for sequencing according to the standard procedures and the type of sequencing technology desired.

·      iDeal ChIP-seq kit for Transcription Factors (Diagenode, Cat. No. C01010054)

Steps 1 to 8 are the same as for the above Histones protocol.

9. Prepare 4 ml of 1x ChIP buffer iC1b by mixing the following reagents:

• 3.2 ml ChIP-seq grade water

• 0.8 ml 5x ChIP buffer iC1b

• 80 µl of 5% BSA Keep the diluted ChIP buffer iC1b on ice.

10. Wash the beads 3 times with 1 ml of ice-cold 1x ChIP buffer iC1b. To wash the beads, add 1 ml of 1x ChIP buffer iC1b directly to the beads suspension, resuspend the beads by pipetting up and down several times and incubate at 4°C with gentle shaking for 5 minutes. Spin the tubes and place them in the 1.5 ml Diagenode magnetic rack. Wait for one minute to allow the beads to be captured by the magnet and remove the supernatant. Repeat this 2 times.

10. After the last wash, resuspend the beads in 1x ChIP buffer iC1b adding the original volume of beads (this means 30 µl per IP).

11. Prepare the ChIP reaction mix as described below for 1 IP:

• 6 µl of BSA

• 1.8 µl 200x protease inhibitor cocktail

• 20 µl 5x iC1b buffer

• 1 µg of ChIP-seq grade antibody

• Up to 70 µl of ChIP-seq grade water

12. Mix 30 µl of the washed beads with 70 µl of ChIP reaction mix per IP. Incubate the tubes for 2-4 hours at 4°C under constant rotation on the DiaMag Rotator.

13. Briefly spin the tubes containing the ChIP reaction mix and add 250 µl of sheared chromatin (typically 15 µg per IP of chromatin). Keep aside 1 µl of the sheared chromatin at 4°C to be used as an input.

14. Perform the washes as follows: briefly spin the tubes and place them in the DiaMag1.5. Wait for one minute and remove the supernatant. Add 350 µl of Wash buffer iW1 and gently shake the tubes to resuspend the beads and incubate for 5 minutes on the DiaMag Rotator at 4°C.

15. Repeat the washing step as described above once with Wash buffer iW2, iW3 and iW4, respectively.

16. Elute the DNA as describe above for the Histones protocol, following steps 15 to 27.

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