title: GSM4790518: MX22; Rattus norvegicus; MeDIP-Seq
library_construction_protocol: For each sample, the epididymis was dissected free of fat and connective tissue, then, after cutting open the cauda, placed into 6 ml of phosphate buffer saline (PBS) for 20 minutes at room temperature. Further incubation at 4ºC will immobilize the sperm.  The tissue was then minced, the released sperm pelleted at 4ºC 3,000 x g for 10 min, then resuspended in NIM buffer and stored at -80ºC for further processing.   DNA protocol: While sperm are thawing, turn on the centrifuge to 4ºC.   1. Re-suspend sperm pellet with pestle (and pestle motor if available).   o If there is some liquid left with the pellet, there is no need to add any additional buffer  o If not add 100 ?l 1xPBS or NIM buffer  2. Take 25 to 50 ?l of the suspension (depending on how big the pellet is) and transfer it to a microfuge tube with 200 ?l 1xPBS.   o Mix  3. Sonicate suspension at 25% with probe sonicator for 5 seconds.  4. Spin at 12,000 rpm for 10 minutes at 4?C.  o While waiting, turn on heat plate to 65?C   5. Discard supernatant. Be careful – pellet is loose.  6. Wash with 200 ?l 1xPBS and spin as above.  o While waiting, retrieve DTT from freezer (make sure there is enough)    o Turn on the incubator  o Vortex DTT once it has thawed a bit  7. Resuspend pellet in 100 ?l 1xPBS, then continue with protocol below.  General:  1. Re-suspend tissue in 100 ?l PBS or NIM buffer, then   o add 820 ?l DNA extraction buffer   o add 50 ?l 0.1M DTT   o vortex briefly  o incubate at 65?C for 15 minutes   o Retrieve ProteinaseK from freezer  2. Add 50 ?l proteinase K (20 mg/ml) and   o incubate at 55?C for 2-3 hours under constant rotation  o use Eppendorf SafeLock tubes and wrap the tops with Parafilm  3. After incubation add 300 ?l of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A)  o mix (vortex) thoroughly and make sure that the solutions are mixed   o then incubate for 20 min on ice (or longer if needed)  4. Centrifuge at 13,500 g for 30 minutes at 4?C.  o Prepare 2 mL tubes  o Take glycoblue out of the freezer  5. Transfer 1 ml of the supernatant to a new 2 ml tube.   o add 2 ?l of glycoblue   o add 1 ml of cold 100 % isopropanol  6. Mix well by inverting tubes several times.  7. Incubate for 60 min at -20?C.  8. Centrifuge for 20 min at 13,500 g at 4?C.   9. Take off and discard supernatant without disturbing (blue) pellet.   10. Wash pellet 2-3 times with 70% cold ethanol.   o Add 500?l of 70% ethanol to each pellet and   o return to freezer for 20 minutes  11. Centrifuge for 10 min at 4?C at maximum speed (18,200 g)   o discard supernatant  12. Spin again briefly to collect residual ethanol at bottom of tube and remove as much liquid as possible with gel loading tip.  13. Air-dry at RT until pellet looks dry, but be careful not to over-dry.   o This will take about 5 minutes  14. Re-suspend in 100 ?l of nuclease free water or TE.  15. Measure DNA concentration using Nanodrop.   Methylated DNA immunoprecipitation protocol: Shear & Add Antibody to DNA  1. Sonicate purified genomic DNA using Covaris:  a. Dilute 6?g genomic DNA into 50 or 130 ?l TE buffer in the appropriate Covaris tube.  b. Set Covaris to desired program (300 or 500 bp program works well).  c. Run program for each tube  • While waiting, turn on the dry bath to 95ºC.   2. Run 10 ?l sonicated DNA & 5 ?l DNA ladder on 2% agarose gel to verify fragment size.  • While waiting for gel, clean up Covaris.   3. Measure volume of sonicated DNA after gel run.  4. Dilute sonicated DNA with TE buffer to 400 ?l (400 – 130 + 10 = 280 µL)  5. Heat-denature for 10min at 95?C, and   • immediately cool on (watery) ice for 5min  6. To the denatured sonicated DNA:  a. Add 100?l of cold 5X IP buffer   b. Add 5?g (volume depending on concentration) of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006). ALWAYS check antibody concentration. Diagenode sometimes changes it.  c. Incubate the DNA-antibody mixture overnight on a rotator at 4?C.  Rotate at a low enough speed to prevent significant foaming.  Bind Beads to DNA: Antibody Mixture  1. Pre-wash Magnetic Beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) as follows:  • Resuspend the beads in the vial (pipet up and down or rotate, do not vortex)  • Transfer desired volume (50 ?l per sample) to a centrifuge tube, add the same volume of Washing Buffer (at least 1 mL), and resuspend.  • Place the tube in a magnet for 1-2 minutes and discard supernatant.   • Resuspend beads in 1 ml of washing buffer and incubate for 1 minute on bench. Put tube on magnet for 1-2 minutes and discard supernatant.  • Remove the tube from the magnet and resuspend the washed beads in the same volume of 1xIP buffer as the initial volume of beads.  2. Add 50?l of beads to the 500?l of DNA-antibody mixture from step 2  3. Incubate 2h on a rotating platform at 4?C.  Wash DNA: Antibody: Beads Mixture & Add Proteinase K Mix  1. Wash beads three times with 1X IP buffer as follows:  • Place tube in magnet for 1-2 minutes and discard supernatant  • Remove tube from magnet  • Add 1ml cold 1X IP buffer  • Mix by inverting tube or gently vortexing (set Vortex to low).  • Incubate tube(s) for 1 minute on bench.  • Place tube in magnet for 1-2 minutes and discard supernatant  • Repeat twice for a total of 3 washes  2. Resuspend the beads in 250?l digestion buffer.  3. Add 3.5?l Proteinase K Mix (20mg/ml) to the resuspended beads.  4. Incubate 2-3 hours on a rotating platform at 55?C (make sure to seal lids so they don't leak). Use USA Scientific SealRite tubes or Eppendorf Safe-Lock tubes and seal top with Parafilm.  Purify DNA  • Retrieve Glycoblue from freezer.   1. Remove Parafilm and add 250?l Phenol-Chloroform-Isoamylalcohol to each tube.    • Vortex for 30 sec and centrifuge at 14,000g for 5min at RT.  • Remove the aqueous supernatant and transfer to a fresh microcentrifuge tube.  2. Add 250?l chloroform to the supernatant from step 1.    • Vortex for 30sec and centrifuge at 14,000g for 5min at RT.   • Remove the aqueous supernatant and transfer to a fresh microcentrifuge tube.  3. Add 500 ?l ethanol  • Then add 20?l 5M NaCl   • Then add 2?l glyco-blue (20mg/ml)   • Vortex well   4. Precipitate in -20?C freezer for 1 hour to overnight.  5. Centrifuge at 14,000g for 20min at 4?C.  Carefully remove the supernatant, while not disturbing the pellet.  6. Wash the pellet with 500?l cold 75% ethanol. Leave in -20?C freezer for 15 min.  7. Centrifuge at 14,000g for 5min at 4?C.  Carefully remove the supernatant.  8. Completely dry samples.  9. Resuspend in 25?l H2O.    10. Measure the DNA concentration in Qubit with ssDNA kit and matching buffer. MeDIP DNA was used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra DNA Library following the manufacturer's protocol  and indexing each sample individually with NEBNext Multiplex Oligos for Illumina. NEBNext Ultra II Library Prep Kit for Illumina: ssDNA  • Start with 20 µl in PCR strips.   o ~40 ng of DNA  o Complete the volume to 20 µl   • Add 2.5 µL of diluted (1:10) random primer to each tube.   o for 10 samples, 27 µL 0.1x TE and 3 µL primers  o for 12 samples 36 µL 0.1x TE and 4 µL primers  o for 22 samples, 54 µL 0.1x TE and 6 µL primers  • Place strips in the thermocycler and use the Denaturation program. Immediately remove (as soon as time runs out on the program) and cool on watery ice following denaturation. Let tubes rest on watery ice for 5 minutes, then transfer to refrigerated block.   I. Second Strand cDNA Synthesis  Assemble the second strand cDNA synthesis reaction on ice by adding the following components to the 20 µl DNA.   Second Strand Synthesis Reaction  DNA          20 µL  ORANGE NEBNet 2nd Strand Synthesis Reaction Buffer  8 µL  ORANGE NEBNext 2nd Strand Synthesis Enzyme Mix 4 µL  Nuclease-free water      45.5 µL  Total Volume        ~70 µL  1. Keeping the tube on ice, mix thoroughly by pipetting t