Illumina HiSeq 2500 sequencing; Sequencing of methylated GATC DNA fragments (Targeted DamID) from seven olfactory neuron populations in Drosophila melanogaster
Sequencing of methylated GATC DNA fragments (Targeted DamID) from seven olfactory neuron populations in Drosophila melanogaster
1. Cross the flies of interest (homozygous and not balanced) with flies UAS LT3-Dam and UAS LT3-Dam Pol II. Set up approx. 40 tubes 2. Extract the antennae with liquid nitrogen using Carol's protocol. Collect the antennae in a 1.5ml eppendorf tube. 3. Aspirate the supernatant and rince once with PBS1x in order to get rid of the triton. 4. Take off all the supernatant possible and put 50µl of PBS. Homogenise with a blue pestle for at least 2 min in order to break the maximum of antennae. 5. Rince the pestle with 100µl PBS and add 40μl 100mM EDTA 6. Add 20μl RNase (12.5μg/μl) (Qiagen #19101) and mix with pipette. Let sit for 3-5 min. 7. Add 20μl Proteinase K (Qiagen DNeasy Blood & Tissue Kit #69504) and 200µl Buffer AL. Gently pipette up and down with blue tip (~50 times) and put at 56°C for 30 min (heat block) 8. Centrifuge 30 sec at 10'000rpm and transfer the supernatant in a new tube  1. Cool to RT, add 200μl 100% Ethanol and mix (gently pipette with blue tip)2. Add all solution to a spin column3. Spin at 13'000 rpm for 1 min and discard the flow-through4. Add 500µl of AW1 solution and spin 13'000rpm for 1 min, discard flow-through and collection tube5. Wash with 500µl of AW2 solution and spin 13'000rpm for 1 min, discard flow-through6. Wash a second time 500µl of AW2 solution and spin 13'000rpm for 3 min, discard flow-through7. Spin 1min to get rid of the maximum of liquid.8. Put the column on a new eppendorf tube, add 200µl of AE Buffer on the column and leave at RT for at least 30 min. Spin at13'000rpm for 1 min and keep the flow-through.9. Add 200µl of AE Buffer and leave at RT for 10 min. Spin at13'000rpm for 1 min and keep the flow-through.10. Combine the 2 elutions (400µl of total volume). 11. Set aside 3-4µl to run on gel to check quality (should be a single band on the top of gel, not a smear). Check concentration on the nanodrop.12. Add 800μl 100% ethanol, 40μl 3M NaAcetate, and 1μl of molecular biology grade glycogen. Mix by inversion and leave at -20°C for at least 30 min.13. Spin at 13'000rpm at 4°C for 30 min, carefully  remove the supernatant (there should be a small visible pellet)14. Add 500μl 70% ethanol, spin 13000rpm at 4°C for 10 min, remove ethanol and open the lid to dry the pellet (gDNA) – don't completely dry the pellet (<5min)15. Add 50µl DEPC H2O and let resuspend the pellet.16. Check concentration on the nanodrop. Dpn I Digestion :  1. Take 4µg of genomic DNA and perform a digestion with Dpn I at 37°C, ON.  (Do not mix, pipette or vortex !!)  Dpn I Digestion Mix : X µl Genomic DNA X µl H2O 5.0 µl Buffer Cutsmart (NEB) 1.5 µl Dpn I (NEB) ----------- 50 µl  2. Add another 0.5µl Dpn I enzyme, mix well with blue tip and leave another hou rat 37°C. 3. Incubate at 80°C heat block for 20 min to inactivate Dpn I. Cool on ice before next step. 4. Clean-up with Qiagen Purification Kit (#28104). Add 250µl of Buffer PB and follow the protocol. Elute in 24µl DEPC H2O. 5. Mesure DNA concentration.    Adaptor Ligation:  1. Make ds AdR stock (50µM) : See Appendix 2. Use 350ng of digested DNA (using a little bit less - like 300ng - is ok too !). Set up ligation reaction with Rapid DNA Ligation Kit (Roche #11 635 379 001) and leave it for 2h at 16°C. 3. Inactivate at 65°C for 10 min.   Ligation Mix :  X  µl  H2O X  µl Digested DNA (maximum volume possible 7.2µl) 0.8 µl AdR primer (100µM) (see Appendix) 2.0 µl  5x Buffer 2 10.0 µl 2x Buffer 1 1.0 µl Ligase ---------- 21 µl      Dpn II Digestion :  1. Perform a Dpn II digestion, directly in the ligation tube (total volume of 80 µl) 2. Digest ON at 37°C  Dpn II Digestion Mix : 21 µl Ligated DNA 50 µl H2O 8.0 µl Buffer Dpn II (NEB) 1.0 µl Dpn II (NEB) ----------- 80 µl   PCR Amplification:  1. Take 40µl from Dpn II digested gDNA, mix with 120µl PCR mix (Advantage cDNA Polymerase Mix, Clontech #639105) and split int 4x 40µl reactions in a PCR strip. 2. Store the remaining 40µl at -20°C for further amplification if required.  PCR Mastermix :  16 µl  10x cDNA PCR reaction buffer 2.5 µl AdR PCR Primer 3.2 µl dNTP mix 10mM 2.0 µl Advantage DNA polymerase mix 96.3 µl DEPC H2O  PCR programme :  68°C – 10 min 94°C – 30sec 65°C – 5 min 68°C – 15 min  Reepeat 3x  94°C – 30 sec  65°C – 1 min  68°C – 10 min  Repeat 17x  94°C – 30 sec  65°C – 1 min  68°C – 10 min  Keep at 4°c  3. Pool the 4x 40µl PCR reaction and run 4µl of it to check quality (should be a smear) 4. Clean-up with Qiagen Purification Kit. Add 750µl of Buffer PB (150µl sample) and follow the protocol. Elute in 32µl DEPC H2O. 5. Mesure DNA concentration. Sonication:  1. Use the COVARIS machine in order to obtain a ~300bp average fragment size. 2. Dilute the samples : 2µg of DNA in 100µl H2O 3. Follow the protocol procedure using the settings to reach an average of 400bp (Target BP400)  COVARIS Protocol : - Fill the tank with distilled water until the mark « 15 » (on the left) and close the lid. - Switch on the machine - Switch on the LTC1 switch under the table (for the cooling down) - Switch on the computer and open « Sonolab » - Push DEGAS botton -> ON (it will take ~45min to cool down to 7°C) - Transfer the samples to the COVARIS tubes ( Microtube AFA Fiber Pre-Slit  Snap cap 6x16mm #520045) - Choose the parameters to reach the average bp wanted (i.e. Target BP400) : Configure -> enter the values.  Intensity : 4  Duty cycle : 10%  Cycles per Burst : 200  Treatment time : 55  (The maximum time is 60 sec, if you need more time do 2 cycles) - Put a tube in the red holder up side down, making sure that it is well placed. Place it in tje machine and press start. - Proceed the same way for all the samples. - At the end, exit the program, shut down computer and machine.  4. Give 2µl to the GTF for Fragment Analysis and Q-Bit quantification. If average size is correct, go on with the digestion.    Sau3AI Digestion :  1. Perform a digestion ON with Sau3AI (98µl sample + 11µl Buffer 1.1 + 1 µl enzyme) to get rid of the adaptors.  2. Clean-Up with Qiagen kit and elute in 32µl. 3. Check sample concentration         Appendix  Ligation of DamID adaptors Making ds AdR stock (50uM): Take 50 μL AdRt (100uM) and 50 μL AdRb (100 uM) Mix together and place into a beaker with water that has just boiled. Let the beaker cool down to RT in order to let the adaptors anneal slowly.  Primer sequences : AdRt CTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAGGA AdRb TCCTCGGCCG   Primer for PCR :  Adr_PCR :   GGTCGCGGCCGAGGATC