16-20 week human fetal brain IPed with Foxp2 antibody
Biomaterial provider
Brain and Tissue Bank for Developmental Disorders at the University of Maryland at Baltimor
Treatment protocol
Cells were crosslinked and sonicated
Growth protocol
Tissue was frozen and stored at -80 degrees C until crosslinking procedure
Extracted molecule
genomic DNA
Extraction protocol
Hypotonic solution was used to isolate DNA and protein crosslinked material Prepare chromatin from frozen cell pellets Materials Cell: two pellet, each from 0.5x 10^9 cells. Lysis buffer 1, 100ml, add protease inhibitor tablet before use. (Zheng uses the protease inhibitors at the end of the protocol-he bought the Sigma protease panel) 5ml 1M Hepes-KOH, pH 7.5 2.8ml 5M NaCl 0.2ml 0.5M EDTA 10ml glycerol 5ml 10% NP-40 0.25ml Triton X-100 add 76.75 ml H2O buffer 2, 100ml, add protease inhibitor tablet before use 4ml 5M NaCl 0.2ml 0.5M EDTA 0.1ml 0.5M EGTA (add .125 of 0.4M) 0.5ml 2M Tris pH 8 add 95.2 H2O buffer 3, 100ml and add protease inhibitor tablet before use. 0.2ml 0.5M EDTA 0.1ml 0.5M EGTA 0.5ml 2M Tris-HCl, pH8 Procedures 1. Resuspend each tube of cells (5x10^8 cells)(zheng only used 2x10^8) in 30ml of Lysis buffer I. Rock at 4°C for 10’. Then spin at 4000rpm in a table top centrifuge, 10’ at 4°C. -check under scope for complete lysis. 2. Resuspend each tube of cells in 24ml of buffer 2. Rock gently at room temperature for 10 min. Pellet nuclei in tabletop centrifuge by spinning at 4K rpm, 10 min at 4°C. 3. Resuspend both pellets from each tube in a total of 4ml buffer 3. Transfer into two 15ml conical tubes. Place the tube into a 50-ml tube with ice in it. 4. Sonicate the suspension in 15ml conical tube with a microtip attached to Branson 450 sonifier, setting at 5, constant power. Sonicate 8-10 times with 25sec, allowing the suspension to cool on ice for 1 min between pulses. Take 40ul of extracts every two cycles. 5. Pool all the lysate together. Add 10mg of sarkosyl (Sigma L-5125) and rock for 10min at room temperature. Transfer to falcon tubes. Spin out debris at 10K, 10min, in a ss34-rotor (13,000 RPM at 4 degrees in microfuge). 6. Proceed to next step (immunoprecipitation) or freeze cell extracts in –80°C as 1ml aliquots. Step 3. Chromatin immunoprecipitation Materials: Cell extracts prepared from step 2. Antibody-coupled magnetic beads from step 0 elution buffer: to make 1 ml 50mM Tris pH8 50 ul 1M Tris 10mM EDTA 20 ul 0.5M EDTA 1% SDS 100 ul 830 ul H2O Procedures: 1. set up IP reactions with crude extract: take 1 ml cell extract; add the following: material cconc. amount added ---------- --------- ---------------- Triton-x100 0.1% 0.01 ml of 10% deoxycholate 0.1% 0.01ml of 10% PMSF 1x 0.01ml of 100x Benz 1x 0.01ml of 100x Approtenin 1x 0.005ml of 200x leup 1x 0.001ml of 1000x
2. Add 100ul of magnetic beads pre-coupled with antibody to the tube. incubate at 4°C overnight in a rotating platform.---Save 40 ul for elution control 3. Use a magnet MPC-E (from Dynal) to precipitate the beads, discard supernatant. In cold room
4. Wash 5 times with 1ml wash buffer, with 3’ incubation on rotating platform in cold room, and aspiration. wash buffer, (RIPA buffer) -------------------- final 100ml 10 ml 50mM Hepes, pH 7.6 5ml of 1M Hepes 0.5ml 1mM EDTA 200ul of 0.5M 20 ul 0.7% DOC 7ml of 10% 0.7 ml 1% NP-40 (IPGEL) 10ml of 10% 1.0 ml 0.5M LiCl 2.12g of powder [sigma] .2 grm 1mM PMSF 1ml of 100x 100 ul leupeptin 1ml of 100x 100 ul aprotinin water 77.8 ml 7.8 ml Add NP-40 first and then DOC to reduce cloudiness. Wash once with 1 ml TE (pH 8.0). After removing the TE by aspiration, spin the tubes for 3 minutes at 3000 rpm and remove any remaining liquid with a pipette.
5. Elution from beads and reversal of cross links Add 50 µl elution buffer, vortex briefly to resuspend the beads and incubate at 65°C for 12 minutes. Vortex lightly every 2 minutes during the incubation. 6. Spin for 30 seconds at maximum speed and transfer 50 µl of supernatant to a new tube. Discard the rest. 7. Add 150 µl of TE/SDS to the supernatant in the new tube in order to reverse the crosslinking reaction. In the meanwhile, take 40ul of cell extract taken in the step 1, add 120ul of TE/SDS. Incubate overnight at 65°C in an incubator.
Brain and Tissue Bank for Developmental Disorders at the University of Maryland at Baltimor
Treatment protocol
total genome control-no IP
Growth protocol
Tissue was frozen and stored at -80 degrees C until crosslinking procedure
Extracted molecule
genomic DNA
Extraction protocol
Hypotonic solution was used to isolate DNA and protein crosslinked material Prepare chromatin from frozen cell pellets Materials Cell: two pellet, each from 0.5x 10^9 cells. Lysis buffer 1, 100ml, add protease inhibitor tablet before use. (Zheng uses the protease inhibitors at the end of the protocol-he bought the Sigma protease panel) 5ml 1M Hepes-KOH, pH 7.5 2.8ml 5M NaCl 0.2ml 0.5M EDTA 10ml glycerol 5ml 10% NP-40 0.25ml Triton X-100 add 76.75 ml H2O buffer 2, 100ml, add protease inhibitor tablet before use 4ml 5M NaCl 0.2ml 0.5M EDTA 0.1ml 0.5M EGTA (add .125 of 0.4M) 0.5ml 2M Tris pH 8 add 95.2 H2O buffer 3, 100ml and add protease inhibitor tablet before use. 0.2ml 0.5M EDTA 0.1ml 0.5M EGTA 0.5ml 2M Tris-HCl, pH8 Procedures 1. Resuspend each tube of cells (5x10^8 cells)(zheng only used 2x10^8) in 30ml of Lysis buffer I. Rock at 4°C for 10’. Then spin at 4000rpm in a table top centrifuge, 10’ at 4°C. -check under scope for complete lysis. 2. Resuspend each tube of cells in 24ml of buffer 2. Rock gently at room temperature for 10 min. Pellet nuclei in tabletop centrifuge by spinning at 4K rpm, 10 min at 4°C. 3. Resuspend both pellets from each tube in a total of 4ml buffer 3. Transfer into two 15ml conical tubes. Place the tube into a 50-ml tube with ice in it. 4. Sonicate the suspension in 15ml conical tube with a microtip attached to Branson 450 sonifier, setting at 5, constant power. Sonicate 8-10 times with 25sec, allowing the suspension to cool on ice for 1 min between pulses. Take 40ul of extracts every two cycles. 5. Pool all the lysate together. Add 10mg of sarkosyl (Sigma L-5125) and rock for 10min at room temperature. Transfer to falcon tubes. Spin out debris at 10K, 10min, in a ss34-rotor (13,000 RPM at 4 degrees in microfuge). 6. Proceed to next step (immunoprecipitation) or freeze cell extracts in –80°C as 1ml aliquots. Step 3. Chromatin immunoprecipitation Materials: Cell extracts prepared from step 2. Antibody-coupled magnetic beads from step 0 elution buffer: to make 1 ml 50mM Tris pH8 50 ul 1M Tris 10mM EDTA 20 ul 0.5M EDTA 1% SDS 100 ul 830 ul H2O Procedures: 1. set up IP reactions with crude extract: take 1 ml cell extract; add the following: material cconc. amount added ---------- --------- ---------------- Triton-x100 0.1% 0.01 ml of 10% deoxycholate 0.1% 0.01ml of 10% PMSF 1x 0.01ml of 100x Benz 1x 0.01ml of 100x Approtenin 1x 0.005ml of 200x leup 1x 0.001ml of 1000x
2. Add 100ul of magnetic beads pre-coupled with antibody to the tube. incubate at 4°C overnight in a rotating platform.---Save 40 ul for elution control 3. Use a magnet MPC-E (from Dynal) to precipitate the beads, discard supernatant. In cold room
4. Wash 5 times with 1ml wash buffer, with 3’ incubation on rotating platform in cold room, and aspiration. wash buffer, (RIPA buffer) -------------------- final 100ml 10 ml 50mM Hepes, pH 7.6 5ml of 1M Hepes 0.5ml 1mM EDTA 200ul of 0.5M 20 ul 0.7% DOC 7ml of 10% 0.7 ml 1% NP-40 (IPGEL) 10ml of 10% 1.0 ml 0.5M LiCl 2.12g of powder [sigma] .2 grm 1mM PMSF 1ml of 100x 100 ul leupeptin 1ml of 100x 100 ul aprotinin water 77.8 ml 7.8 ml Add NP-40 first and then DOC to reduce cloudiness. Wash once with 1 ml TE (pH 8.0). After removing the TE by aspiration, spin the tubes for 3 minutes at 3000 rpm and remove any remaining liquid with a pipette.
5. Elution from beads and reversal of cross links Add 50 µl elution buffer, vortex briefly to resuspend the beads and incubate at 65°C for 12 minutes. Vortex lightly every 2 minutes during the incubation. 6. Spin for 30 seconds at maximum speed and transfer 50 µl of supernatant to a new tube. Discard the rest. 7. Add 150 µl of TE/SDS to the supernatant in the new tube in order to reverse the crosslinking reaction. In the meanwhile, take 40ul of cell extract taken in the step 1, add 120ul of TE/SDS. Incubate overnight at 65°C in an incubator.
Label
Cy3
Label protocol
Random prime labeling
Hybridization protocol
Previous step: LM-PCR and labeling Materials: hyb buffer: 70% formamide/3XSSC/14.3% dextran sulfate Make this a few hours ahead of time to allow the dextran sulfate to go into solution. To make 10 ml: 1.43 grm dextran sulfate 1.5 ml of 20xSSC 7 ml formamide Bring up to 10 ml with water. -2% of BSA: 1 g of BSA in 50 ml of Mili-Q water -Array Pre-hybridization Solution 2XSSC/0.05%SDS/0.2%BSA. (to make take premade solution and add 0.16 grm of BSA to 80 ml solution) -DNA: purified from random labeling step. -Cot-1 DNA (from Invitrogen), need to measure DNA concentration for each tube. Do not trust the concentration of company label. -One water bath at 42 degrees -One water bath at 60 degrees -Heat block at 95 degrees -Incubator at 37 degrees Procedures: A. Array Pre-hybridization 1. Fresh made pre-hybridization solution: 2X SSC/0.05% SDS/0.2% BSA (use fresh made BSA). 2. Add 80 ml of pre-hyb solution into a Coplin jar and warm up solution to 42°C in water bath (take about 20-30 min). 3. Soak slides in the Coplin jar (max. 4 slides/jar) and incubate for 40 min, at 42°C. Then rinse with RO water for 10 sec, then spin at 2k rpm in epperdurf 5840 for 30”). Place in Corning Hyb chamber. B. Preparing the Hybridization Solution In the meanwhile, Mix 2.0ug Cy3 labeled DNA and 2.0ug of Cy5 labeled DNA, add 36ug of human Cot-1 DNA, and dH2O so that final volume is 130ul. Add 14ul 3M NaOAc, 280ul EtOH. Mix by vortexing, freeze in dry ice for 15’, then spin 15’ at 14k in cold room. Wash 1x, and dry in air. Dissolve the pellet in 22.4 ul of buffer (2.2x ssc, 0.22% SDS), incubate at 37°C for 10 min to dissolve. Resuspend by pipeting. Add 20 µl Hybridization buffer. Denature probe at 95 C for 5 min, then spin for 30 seconds, Incubate at 42°C for 2 minutes. Add 4ul yeast tRNA (at 10ug/ul). Add 3.0ul of 2% BSA. Incubate at 42°C for 5 min. Spin briefly. C. Hybridization: spot 47ul to the array in three evenly spaced droplets. Place cover slip on and incubate at 60°C for overnight. (16 hours at least). Step 10---washing materials: 400ml 2x SSC, 0.1% SDS, preheated to 60°C 300ml 0.1x SSC, 0.1% SDS 1 liter of 0.1X SSC. Procedures: Note: Although the 2X SSC solutions are preheated to 60°C the washes are actually done at room temperature. 1. Fill a Coplin jar with 50ml 2X SSC, 0.1% SDS pre-heated to 60ºC. Gently place the array into the jar, barcode up. Let the Gene Array sit for 1 minute then remove each array slowly from the Coplin jar leaving the cover slip behind. 2. Place the array into a slide rack submersed in 2X SSC, 0.1% SDS pre-heated to 42ºC in a Wheaton glass staining dish. Incubate the array for 5 minutes with gentle agitation (rocking on the rocker platform). 3. Remove the slide rack from the 2X SSC, 0.1% SDS solution and place in 250ml of room temperature 0.1X SSC, 0.1% SDS in a Wheaton glass staining dish. Incubate for 10 minutes with gentle agitation. 4. Remove the slide rack from the 0.1x SSC, 0.1% SDS solution and place in 250ml of room temperature 0.1X SSC in a Wheaton glass staining dish for 15 seconds. Agitate gently by hand. This quick wash removes much of the residual SDS. 5. Remove the slide rack from the 250ml of 0.1X SSC solution and place it in 250ml of 0.1X SSC in a glass staining dish. Incubate for 1 minutes (with gentle agitation if desired). 6. Repeat step 5 two more times (3 total) 7. After 15 seconds remove the array slowly from the slide rack and place into the rack on centrifuge. Balance well. Spin for 1 minute at 1500rpm in eppendurf 5840. After spin, store slides in slide boxes. Protect slides from dust and light until analyzed.
Scan protocol
Arrays were scanned on a GenePix 4000B (Molecular Devices, Sunnyvale, CA) and image analysis was performed with GenePix Pro 6.0.
Description
n/a
Data processing
Normalized ratios were obtained subtracting the log2-transformed
expression value in the control sample from the log2-transformed
expression value in the experimental sample, after median normalization.
